Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsapm.4c00486

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DOI： 10.1021/acsanm.4c01103

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Methaemoglobin (metHb) possesses inherent characteristics that facilitate reversible binding to hydrogen sulfide. Exogenous hydrogen sulfide supplementation imparts beneficial bioactive effects, including antioxidant and anti-inflammatory; hence, we hypothesized that the metHb-hydrogen...

DOI： 10.1039/d4tb01621a

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DOI： 10.1021/acsabm.3c00888

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DOI： 10.1016/j.brainres.2023.148592

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Photodynamic therapy (PDT) efficiently induces apoptosis through visible‐light irradiation of photosensitizers (PSs) within tumors and microbial cells. Porphyrin analogues serve as widely utilized photosensitizing agents with their theranostic abilities being governed by molecular structures and central metal ions. However, these macrocyclic compounds tend to agglutinate and form stacks in aqueous environments, resulting in a loss of photochemical activity. To overcome this limitation, encapsulation within liposomes and polymer micelles enables the dispersion of porphyrins as monomolecular entities in aqueous solutions, preventing undesirable deactivation. Recently, the use reconstituted hemoproteins containing various metal‐porphyrins and protein cages incorporating porphyrins have garnered significant interest as a new generation of biocompatible PSs. In this concept paper, we provide a comprehensive review of recent developments and trends of protein–porphyrin complex PSs for applications in anticancer and antimicrobial PDTs.

DOI： 10.1002/cmdc.202300373

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DOI： 10.1016/j.ijpharm.2023.123433

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Hemoglobin wrapped covalently with poly(2-ethyl-2-oxazoline)s (POx-Hb) is characterized physicochemically and physiologically as an artificial O2 carrier for use as a red blood cell (RBC) substitute. The POx-Hb is generated by linkage of porcine Hb surface-lysines to a sulfhydryl terminus of the POx derivative, with the average binding number of the polymers ascertained as 6. The POx-Hb shows moderately higher colloid osmotic activity and O2 affinity than the naked Hb. Human adult HbA conjugated with POx also possesses equivalent features and O2 binding properties. The POx-Hb solution exhibits good hemocompatibility, with no influence on the functions of platelets, granulocytes, and monocytes. Its circulation half-life in rats is 14 times longer than that of naked Hb. Hemorrhagic shock in rats is relieved sufficiently by infusion of the POx-Hb solution, as revealed by improvements of circulatory parameters. Serum biochemistry tests and histopathological observations indicate no acute toxicity or abnormality in the related organs. All results indicate that POx-Hb represents an attractive alternative for RBCs and a useful O2 therapeutic reagent in transfusion medicine.

DOI： 10.1021/acsabm.3c00392

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DOI： 10.1021/acsomega.3c01054

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DOI： 10.1038/s41598-023-35999-4

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

DOI： 10.1246/cl.230105

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Abstract

Myoglobin combined with human serum albumin (Mb‐HSA) can be produced using yeast Pichia pastoris as a host strain, with secretion into the culture medium. This Mb‐HSA fusion protein possesses identical O₂ binding affinity to that of naked Mb. The Mb unit is reconstituted with a zinc(II) protoporphyrin IX, yielding (zinc substituted Mb)‐HSA, ZnMb‐HSA. The photophysical property and singlet O₂ generation ability of ZnMb‐HSA are equivalent to those of ZnMb. In vitro cell experiments revealed that ZnMb‐HSA acts as a superior photosensitizer for photodynamic cancer therapy. It is noteworthy that ZnMb‐HSA shows long circulation lifetime in vivo.

DOI： 10.1002/chem.202203952

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This paper describes the synthesis and O2 binding properties of core-shell structured hemoglobin (Hb) nanoparticles (NPs), artificial O2 carriers of five types, as designed for use as red blood cell (RBC) substitutes. Human adult Hbs were polymerized using α-succinimidyl-ω-maleimide and dithiothreitol in spheroidal shapes to create parent particles. Subsequent covalent wrapping of the sphere with human serum albumin (HSA) yielded 100 nm-diameter Hb nanoparticles (HbNPs). The HbNP showed higher O2 affinity than that of RBC, but NPs prepared under a N2 atmosphere exhibited low O2 affinity. Entirely synthetic particles comprising recombinant human adult Hb and recombinant HSA were also fabricated. Using a recombinant Hb (rHb) variant in which Leu-β28 of the heme pocket had been replaced with Phe, we found somewhat low O2 affinity of rHb(βL28F)NP. Particles made of stroma-free Hb (SFHb) containing natural antioxidant enzyme catalase (SFHbNP) formed a very stable O2 complex, even in aqueous H2O2 solution. The SFHbNP showed good blood compatibility and did not affect the blood cell component functionality. The circulation half-life of SFHbNP in rats was considerably longer than that of naked Hb. All results indicate these Hb-based NPs as useful alternative materials for RBC and as a useful O2 therapeutic reagent in diverse medical scenarios.

DOI： 10.1021/acsabm.2c00813

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Swimming protein microtube motors with an exterior surface of fetuin and an interior surface of catalase captured virus-shaped fluorescent nanoparticles (100 nm-diameter), non-infectious fake influenza A viruses, efficiently.

DOI： 10.1039/d2ma00566b

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.taap.2023.116472

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Catalase enwrapped covalently with human serum albumin incorporating protoporphyrin IX is a unique dual functional hemoprotein cluster (O₂ generation and photosensitization) that can achieve enhanced photodynamic therapy.

DOI： 10.1039/D2MA00242F

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A bovine hemoglobin (HbBv) or human adult hemoglobin (HbA) wrapped covalently by human serum albumins (HSAs), hemoglobin-albumin clusters (HbBv-HSA3 and HbA-HSA3 ), are artificial O2 carriers used as a red blood cell substitute. This article describes the physicochemical properties of the HbBv-HSA3 and HbA-HSA3 solutions, and their abilities to restore the systemic condition after resuscitation from hemorrhagic shock in anesthetized rats. The HbBv-HSA3 and HbA-HSA3 , which have high colloid osmotic activity, showed equivalent solution characteristics and O2 binding parameters. Shock was induced by 50% blood withdrawal. Rats exhibited hypotension and significant metabolic acidosis. After 15 min, the rats were administered shed autologous blood (SAB), HbBv-HSA3 , HbA-HSA3 , or Ringer's lactate (RL) solution. Survival rates, circulation parameters, hematological parameters, and blood gas parameters were monitored during the hemorrhagic shock and for 6 h after administration. All rats in the SAB, HbBv-HSA3 , and HbA-HSA3 groups survived for 6 h. The HbBv-HSA3 and HbA-HSA3 groups restored mean arterial pressure after the resuscitation. No remarkable difference was observed in the time courses of blood gas parameters in any resuscitated group except for the RL group. Serum biochemical tests showed increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the HbBv-HSA3 and HbA-HSA3 groups compared to the SAB group. Therefore, we observed other rats awakened after resuscitation with HbA-HSA3 for 7 days. The blood cell count, AST, and ALT recovered to the baseline values by 7 days. All the results implied that HbBv-HSA3 and HbA-HSA3 clusters provide restoration from hemorrhagic shock as an alternative material for SAB transfusion.

DOI： 10.1002/jbm.b.35040

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DOI： 10.1246/cl.210549

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We report the synthesis of catalase-driven protein microtube motors with different exterior surfaces. Their abilities of bacteria capture, reaction enhancement by self-stirring, and velocity control with light irradiation were highlighted.

DOI： 10.1039/d1ma00610j

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Genetically and chemically tuned haemoglobin–albumin trimers demonstrated haemoglobin allostery and moderately low O₂ affinity, resulting in a higher O₂ transport efficiency compared to red blood cells.

DOI： 10.1039/d1cc03684j

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DOI： 10.1002/cbic.202100213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ajoc.202100184

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/ajoc.202100184

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The first boron complexes of porphycenes, structural isomers of porphyrin, are reported. They are synthesized in good yields by reacting the free-base porphycene ligands with BF ·Et O through a microwave-assisted method. Depending on the substituent group of porphycenes, two different coordination structures, mono- and diboron porphycenes, are obtained simultaneously. The single crystal structures and DFT calculations suggest that the boron atom of the monoboron porphycene is favorably coordinated on the dipyrroethene site, and the regioisomer of diboron porphycene is of cisoid stereochemistry, which is more stable than transoid. We also investigate the protonation behavior of boron porphycene complexes. Diboron porphycene does not undergo protonation, whereas monoboron porphycene undergoes protonation at the nonboron coordinating pyrroline site, resulting in a red shift in both absorption and emission spectra. Protonation and deprotonation of monoboron porphycene can be reversibly triggered using acids and bases. 3 2

DOI： 10.1021/acs.inorgchem.0c01266

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DOI： 10.1039/D0CC07167F

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Language：English   Publishing type：Research paper (scientific journal)  

Regional cold perfusion and hemoglobin-based oxygen carrier administration both exert neuroprotective effects against cerebral ischemia reperfusion injury. We herein investigated whether the combination of these two therapies leads to stronger neuroprotective effects. Combination therapy was performed with the regional perfusion of cold HemoAct, a core-shell structured hemoglobin-albumin cluster, in a rat transient middle cerebral artery occlusion model. The effects of combination therapy, the intra-arterial administration of 10 °C HemoAct (10H) initiated at the onset of reperfusion, were compared with those of monotherapies, the intra-arterial administration of 10 °C saline (10S) and 37 °C HemoAct (37H), and an untreated control under the condition of 2-hour ischemia/24-hour reperfusion. The durability of therapeutic effects and the therapeutic time window of combination therapy were assessed based on comparisons with the 10H and control groups. Significantly better neurological findings and smaller infarct volumes were observed in the three treated (10S, 37H, and 10H) groups than in the control group. Among the 3 treated groups, only the 10H group showed significant improvements over the control group in the other items examined, including cerebral blood flow reduction, brain edema, and protein extravasation. The significant therapeutic effects of combination therapy on neurological disabilities and infarct volumes were confirmed at least until 7 days after reperfusion. Furthermore, combination therapy ameliorated neurological disabilities and hemorrhagic transformation in rats subjected to 4- and 5-hour ischemia/24-hour reperfusion. Since therapeutic effects may be expected until at least 5 h of complete ischemia and reperfusion, this combination therapy is a promising neuroprotective strategy against severe ischemic stroke.

DOI： 10.1016/j.brainres.2020.147012

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DOI： 10.1246/cl.200433

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Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

<p>Recombinant haemoglobin [rHb(βK120C)] was coupled with two human serum albumins (HSAs), yielding a rHb(βK120C)–HSA₂ heterotrimer, which shows a sigmoidal O₂ equilibrium curve and sufficient Hb allostery identical to those of native Hb.</p>

DOI： 10.1039/d0cb00056f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Core-shell protein cluster comprising a hemoglobin (Hb) in the center and human serum albumins (HSA) at the exterior, Hb-HSA3 cluster, is an artificial O2-carrier designed for use as a red blood cell (RBC) substitute. Lyophilization of the Hb-HSA3 cluster solution ([Hb unit] = 5 g/dL) with sucrose and trehalose yielded stable pink powder, which can be stored for 2 years at 4°C. Addition of pure water to the powder regenerated the homogeneous solution. The O2-binding properties and oxyHb rate have been unaltered during the storage period. Infrared spectroscopic studies revealed that the hydrogen bond between protein and sugar stabilizes three-dimensional structure of the cluster.

DOI： 10.1002/pat.4846

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DOI： 10.1039/C9TB02184A

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DOI： 10.1002/asia.201900927

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DOI： 10.1021/acsanm.9b00850

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Previously, we reported on the high-affinity binding of aripiprazole (ARP), an antipsychotic drug, to human albumin and the role of the chlorine atom of ARP on this binding. In this study, we investigated the binding mode of ARP to human albumin in detail using ARP derivatives and several animal-derived albumins. ARP bound strongly to human and dog albumin. The circular dichroism (CD) spectra of ARP bound to human and dog albumin were also similar. Deschloro-ARP bound less strongly to all of the albumin species compared to ARP, and the shapes of CD spectra were similar for all albumin species. CD spectra of dimethyl-ARP, for which chlorine atoms were substituted methyl groups, were quite similar to that of deschloro-ARP. In displacement experiments, competitive binding was observed between ARP and deschloro-ARP. These results suggest that the chlorine atoms in ARP are involved in the binding modes of ARP for human and dog albumins, whereas ARP and deschloro-ARP appear to share the same binding region in site II. The aforementioned results imply that compounds having a chlorine atom bind more strongly to plasma proteins, resulting in a long blood retention time. Therefore, findings reported here may provide the basically useful data for drug design.

DOI： 10.1016/j.xphs.2018.11.045

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DOI： 10.1021/acsomega.8b03474

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japan Society of Microgravity Application  

Serum albumin is the most prominent plasma protein in the blood circulatory system and is the main transporter of various compounds. Although ligand binding ability and crystal structure of human serum albumin (HSA) have been widely studied, there are only few reports on serum albumins of companion animals, particularly dogs and cats. This review describes the synthesis, physicochemical properties, and crystal structures of recombinant canine serum albumin (rCSA) and feline serum albumin (rFSA). The rCSA and rFSA were produced in a gram quantity by genetic engineering procedure using Pichia yeast. The proteins show identical features to those of the native CSA and FSA derived from canine and feline plasma. Single crystals of rFSA were prepared under a microgravity environment in the International Space Station. The overall structures of rCSA and rFSA closely resemble to that of HSA. These recombinant serum albumins are anticipated for use as a therapeutic reagent that can be exploited in numerous veterinary medicine situations.

DOI： 10.15011//jasma.36.360104

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DOI： 10.1021/acs.jpcb.8b10077

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A core-shell protein cluster comprising hemoglobin and human serum albumins, hemoglobin-albumin cluster (Hb-HSA3), was designed and synthesized for use as an artificial O2 carrier and red blood cell (RBC) substitute. For initial preclinical safety evaluation of the Hb-HSA3 solution, we observed blood compatibility in vitro, physiological responses after exchange transfusion into rats and blood circulation lifetime in dogs. Dilution of human whole blood with Hb-HSA3 showed an appropriate decrease in blood cell number, proportional to the mixing volume ratio. Time courses in the circulation parameters and blood gas parameters after 20% exchange transfusion with Hb-HSA3 in anesthetized rats were almost identical to those observed in a sham group (without infusion) and an HSA group (with HSA administration) for 6 h. Serum biochemical tests of the withdrawn blood indicated safety of the protein cluster. Furthermore, fluorescent Hb-HSA3 was infused into beagle dogs to assess blood retention. Fluorescence measurements of the blood samples enabled us to ascertain the cluster half-life within the intravascular space. Histopathologic inspections of the vital organs imply no abnormality in tissues. All these results indicate sufficient initial preclinical safety of Hb-HSA3 as an alternative material for use in RBC transfusion.

DOI： 10.1080/21691401.2018.1505740

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This report describes the synthesis and structure of core-shell protein clusters comprising haemoglobin (Hb) at the centre and recombinant feline serum albumin (rFSA) at the exterior, named as haemoglobin-albumin clusters (Hb-rFSA3). Specifically, we highlight their capability as an artificial O2 carrier that can be used as a red blood cell (RBC) substitute for cats, the most populous pet animal in the world. First, rFSA was expressed by genetic engineering using Pichia yeast. The proteins show identical features to the native FSA derived from feline plasma. Single crystals of rFSA were prepared under a microgravity environment on the international space station (ISS), from which the structure was first revealed at 3.4 Å resolution. Subsequently, bovine Hb was wrapped covalently by rFSA using an α-succinimidyl-ϵ-maleimide crosslinker, yielding Hb-rFSA3 clusters. Three rFSA entities enfolded the Hb nuclei satisfactorily, giving the protein clusters a negative surface net charge (pI = 4.7) and preventing an immunological response against anti-Hb antibodies. The O2 affinity was higher (P50 = 9 Torr) than that of the native Hb. The Hb-rFSA3 clusters are anticipated for use as an alternative material for RBC transfusion, and as an O2 therapeutic reagent that can be exploited in various veterinary medicine scenarios.

DOI： 10.1039/c8tb00211h

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A hemoglobin (Hb) wrapped covalently by three human serum albumins (HSAs) is a triangular protein cluster designed as an artificial O-2-carrier and red blood cell substitute. We report the structural insights into this Hb-HSA(3) cluster in aqueous medium revealed by 3D reconstruction based on cryogenic transmission electron microscopy (cryoTEM) data and small-angle X-ray scattering (SAXS) measurements. Cryo-TEM observations showed individual particles with approximately 15 nm diameter in the vitrified ice layer. Subsequent image processing and 3D reconstruction proved the expected spatial arrangements of an Hb in the center and three HSAs at the periphery. SAXS measurements demonstrated the monodispersity of the Hb-HSA(3) cluster having a molecular mass of 270 kDa. The pair-distance distribution function suggested the existence of oblate-like particles with a maximum dimeter of similar to 17 nm. The supramolecular 3D structure reconstructed from the SAXS intensity using an ab initio procedure was similar to that obtained from cryo-TEM data.

DOI： 10.1021/acs.jpclett.6b02907

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We report the synthesis, structure, and influenza A virus trapping capability of protein nanotubes with an internal wall of fetuin glycoprotein. The nanotubes were fabricated via the alternating layer-by-layer assembly of human serum albumin (HSA), poly-L-arginine (PLA), and fetuin into a track-etched polycarbonate (PC) membrane (pore size, 800 nm), followed by dissolution of the PC template. In an aqueous medium, the (PLA/HSA)5 PLA/fetuin nanotubes captured influenza A virus PR8 (H1N1) efficiently, as revealed by ELISA measurements.

DOI： 10.1246/cl.160805

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There is no blood bank for pet animals. Consequently, veterinarians themselves must obtain "blood" for transfusion therapy. Among the blood components, serum albumin and red blood cells (RBCs) are particularly important to save lives. This paper reports the synthesis, structure, and properties of artificial blood for the exclusive use of dogs. First, recombinant canine serum albumin (rCSA) was produced using genetic engineering with Pichia yeast. The proteins showed identical features to those of the native A derived from canine plasma. Furthermore, we ascertained the crystal structure of rCSA at 3.2 angstrom resolution. Pure rCSA can be used widely for numerous clinical and pharmaceutical applications. Second, hemoglobin wrapped covalently with rCSA, hemoglobin-albumin cluster (Hb-rCSA(3)), was synthesized as an artificial O-2-carrier for the RBC substitute. This cluster possesses satisfactorily negative surface net charge (pI = 4.7), which supports enfolding of the Hb core by rCSA shells. The anti-CSA antibody recognized the rCSA exterior quantitatively. The O-2-binding affinity was high (P-50 = 9 Torr) compared to that of the native Hb. The Hb-rCSA(3) cluster is anticipated for use as an alternative material for RBC transfusion, and as an O-2 therapeutic reagent that can be exploited in various veterinary medicine situations.

DOI： 10.1038/srep36782

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A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA(3) cluster, is an artificial O-2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O-2-binding properties of new hemoglobin. albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA(4), large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA(3), high O-2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA(4) diameter is greater than that of either Hb-HSA(3) or XLHb-HSA(3). The XLHb-HSA(3) showed moderately high O-2-affinity compared to the others because of the chemical linkage between the Cys-93(beta) residues in Hb. Furthermore, the blood circulation behavior of I-125-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA(3) was metabolized faster than Hb-HSA(3) and Hb-HSA(4). Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

DOI： 10.1371/journal.pone.0149526

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Protein nanotubes having an inner surface wall of Candida antarctica lipase B were synthesized by alternating layer-by-layer assembly in a porous polycarbonate membrane. Scanning electron microscopy measurements revealed the formation of uniform hollow cylinders with an outer diameter of 393 +/- 9 nm. Enzymatic ring-opening oligomerization of 12-dodecanolactone was achieved in the one-dimensional pore space interior of nanotube in aqueous medium.

DOI： 10.1246/cl.150789

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

We describe a unique UV-visible absorption spectral property of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (THPP) in the presence of organophosphorus (OP) pesticides. Upon titrating each 16 among total 40 different OP pesticides, the Soret band was significantly red-shifted, and a very intense Q band appeared. They were attributed to the diprotonation of THPP. A suitable solvent for this reaction was determined to be methanol. THPP would become a potential sensor molecule used to detect OP pesticides with high sensitivity in the concentration range of 10(-6) - 10(-4) M.

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This report describes the synthesis, structure, and O-2-binding properties of core-shell clusters composed of human haemoglobin A (HbA) in the centre and human serum albumin (HSA) at the periphery (HbA-HSAm) as potential O-2-carriers designed as red blood cell (RBC) substitutes. Protein clusters were prepared by covalent linkages between HbA lysins and HSA cysteine-34 using a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Major products (m = 2, 3, 4) were isolated using gel filtration chromatography. The low isoelectric points (pI = 5.0-5.2) of the clusters were almost identical to that of HSA, proving the wrapping of HbA by negatively charged HSA. The 3D reconstruction of HbA-HSA(3) based on transmission electron microscopy images revealed a complete triangular structure. The proposed geometries enabled us to assign a possible spatial arrangement of the HbA centre and HSA periphery. The HbA-HSAm clusters showed higher O-2-affinities (P-50 = 8-9 Torr) than the native HbA. The clusters prepared under N-2 atmosphere showed a low O-2-affinity (P-50 = 26 Torr) resembling those of human RBC. Moreover, the cluster containing an alpha alpha-cross-linked HbA with bis(3,5-dibromosalicyl) fumarate showed a markedly low O-2-affinity (P-50 = 35 Torr). These HbA-HSAm clusters with various O-2-affinities can support a new generation of RBC substitutes, which can be better tuned to play a role in O-2 transport.

DOI： 10.1039/c5tb00540j

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A hemoglobin (Hb) wrapped covalently by human serum albumins (HSAs), a core-shell structured hemoglobin-albumin cluster designated as "HemoAct", is an O-2-carrier designed for use as a red blood cell (RBC) substitute. This report describes the blood compatibility, hemodynamic response, and pharmacokinetic properties of HemoAct, and then explains its preclinical safety. Viscosity and blood cell counting measurements revealed that HemoAct has good compatibility with whole blood. Intravenous administration of HemoAct into anesthetized rats elicited no unfavorable increase in systemic blood pressure by vasoconstriction. The half-life of I-125-labeled HemoAct in circulating blood is markedly longer than that of HSA. Serum biochemical tests conducted 7 days after HemoAct infusion yielded equivalent values to those observed in the control group with HSA. Histopathologic inspections of the vital organs revealed no marked abnormality in their tissues. All results indicate that HemoAct has sufficient preclinical safety as an alternative material for RBC transfusion.

DOI： 10.1038/srep12778

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We describe the synthesis of a haemoglobin (Hb) wrapped covalently by recombinant human serum albumin mutants [HSA(Y161H)] containing Mn(III) protoporphyrin IX (MnPP), the Hb-[HSA(Y161H)-MnPP](3) cluster, highlighting the formation of its O-2-complex stable even in H2O2 solution.

DOI： 10.1039/c4cc06076h

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This paper describes the synthesis and structure of protein nanotube (NT) with a lectin interior surface and its size-dependent dextran loading ability in aqueous medium. The NTs were prepared using an alternating layer-by-layer build-up assembly of poly-l-arginine (PLA) and human serum albumin (HSA) in a track-etched polycarbonate (PC) membrane (pore diameter, 400nm), subsequently coating concanavalin A (ConA) as the last layer. Dissolution of the PC template yielded (PLA/HSA)(2)PLA/ConA NTs with 419 +/- 14nm outer diameter and 50 +/- 7nm wall thickness. In a 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffered solution, the NTs captured fluorescein isothiocyanate (FITC)-labeled dextran [molecular weight 4kDa, FITC-Dex(4k)] efficiently into the pores. The ratio of the bound FITC-Dex(4kDa)/ConA was estimated to be 2.1 (mol/mol). Two of four glucosyl-residue binding sites of ConA on the wall presumably faced to the aqueous inner phase of the tube, and they can bind FITC-Dex(4k). On the one hand, only half amount of FITC-Dex was loaded into the channels when the molecular weight of the dextran is greater than 20kDa. Small-angle X-ray scattering measurements revealed that the radius of gyration (R-g) of the FITC-Dex(4k) is 1.45nm (5.0mg/ml), which is satisfactorily small to interact with the each binding site of ConA independently. In contrast, the R-g values of FITC-Dex(20k) and FITC-Dex(40k) were 3.75nm (5.0mg/ml) and 6.62nm (4.0mg/ml), respectively. These large dextrans probably formed an equivalent complex with ConA on the tube wall. Copyright (c) 2014 John Wiley & Sons, Ltd.

DOI： 10.1002/pat.3299

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A covalent core-shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSA(m), is an artificial O-2 carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA(3) cluster with antioxidant activities and its O-2 complex stable in aqueous H2O2 solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1x10(7) M-1, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O-2(center dot-)) and hydrogen peroxide (H2O2), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA(3) captured PtNP into the external albumin unit (K = 1.1x10(7) M-1), yielding an Hb-HSA(3)(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O-2 binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O-2 complex, even in H2O2 solution.

DOI： 10.1371/journal.pone.0110541

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We describe the template synthesis of human serum albumin microtubes (MTs) and highlight their Escherichia coli (E. coli) trapping capability with extremely high efficiency. The E. coli was loaded into the one-dimensional pore space interior of the tubule. Similar MTs including an Fe3O4 layer also captured E. coli and were manipulated by exposure to a magnetic field.

DOI： 10.1039/c4cc03632h

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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A pair of meso-tetrakis {alpha,alpha,alpha,alpha-o-(isonicotinoylamino)phenyl}porphyrins (P1) was coupled by quadruple Pd(II) coordinations, generating a cofacial and fluorescent porphyrin dimer. Photoinduced electron transfers from the dimer to benzoquinones were investigated. Furthermore, Pd(II) coordinations to the alpha,beta,alpha,beta-atropisomer P2 yielded fluorescent porphyrin fiber with a monomolecular width.

DOI： 10.1246/cl.140243

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Genetically engineered human serum albumin (HSA) mutants complexed Mn(iii) protoporphyrin IX (MnPP) in the haem pocket showed superoxide dismutase activity. Replacement of a proximal Tyr-161 by non-coordinating Leu caused a remarkable increase in enzyme activity. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3dt51418h

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We describe the synthesis, structure, and catalytic activity of human serum albumin (HSA) nanotubes (NTs) including gold nanoparticles (AuNPs) as a layered wall component. The NTs were fabricated as an alternating layer-by-layer assembly of AuNP and HSA admixture (a negatively charged part) and poly-L-arginine (PLA, a positively charged part) into a track-etched polycarbonate membrane (400 nm pore diameter) with subsequent dissolution of the template. SEM images showed the formation of uniform hollow cylinders of (PLA/AuNP-HSA)(3) with a 426 +/- 12 nm outer diameter and 65 +/- 7 nm wall thickness. Transmission electron microscopy and energy dispersive X-ray measurements revealed high loading of AuNPs in the tubular wall. HSAs bind strongly onto the individual AuNP (K = 1.25 X 10(9) M-1), generating a core-shell AuNP-HSA corona, which is the requirement of the robust NT formation. Calcination of the (PLA/AuNP-HSA)(3) NTs at 500 degrees C under air yielded red solid NTs composed of thermally fused AuNPs. From the mass decrease by heat treatment, we calculated the weight of the organic components (PLA and HSA) and thereby constructed a six-layer model of the tube. The (PLA/AuNP-HSA)(3) NTs serve as a heterogeneous catalyst for reduction of 4-nitrophenol with sodium borohydrate. Furthermore, implantation of the stiff (PLA/AuNP-HSA)(3) NTs vertically onto glass plate produced uniformly cylindrical tube arrays.

DOI： 10.1021/la403283x

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Covalent core-shell structured protein clusters of hemoglobin (Hb) and human serum albumin (HSA) (HbX-HSA(m)) (m = 2, 3) with novel physiological properties were generated by linkage of Hb surface lysins to HSA cysteine-34 via an alpha-succinimidyl-epsilon-maleimide cross-linker (X: 1 or 2). The isoelectric points of HbX-HSA(m) (pI = 5.0-5.2) were markedly lower than that of Mb and almost identical to that of HSA. AFM and TEM measurements revealed a triangular Hb1-HSA(3) cluster in aqueous medium. The complete 3D structure of Hb1-HSA(3) based on TEM data was reconstructed, revealing two possible conformer variants. All HbX-HSA(m) clusters showed a moderately higher O-2 affinity than the native Hb. Furthermore, the exterior HSA units possess a remarkable ability to bind lumiflavin (LF). The addition of NADH to an aqueous solution of the met-Hb2-(HSA-LF)(3) cluster reduced the inactive ferric Mb center to the functional ferrous Hb. This O-2-carrying hemoprotein cluster with strongly negative surface net charge, high O-2 affinity, and NADH-dependent reductase unit can support a new generation of molecular architecture for red blood cell substitutes.

DOI： 10.1021/bm400204y

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We report structure and photocatalytic activity of solid nanotubes comprising iron oxide (hematite, α-Fe2O3) nanoparticles. The initial precursor cylinders were prepared by alternating layer-by-layer assembly of poly-l-arginine (PLA) and iron-storage protein ferritin in a track-etched polycarbonate membrane (pore diameter: 400 nm) with subsequent dissolution of the template. The obtained (PLA/ferritin)3 nanotubes (outer diameter: 410 ± 14 nm) were calcinated at 500 or 700 °C under air, yielding iron oxide nanotubes. After the calcination, the cylindrical hollow structure completely remained, but its diameter, wall thickness, and maximum length were significantly diminished. SEM measurements revealed that the nanotubes prepared at 500 °C consist of uniform hematite nanoparticles with ca. 5 nm diameter and the nanotubes calcinated at 700 °C are composed of ca. 20 nm hematite nanoparticles. These nanotubes showed efficient photocatalytic activity for degradation of 4-chlorophenol
higher catalytic activity was observed in the reaction with 5 nm hematite nanoparticle nanotubes. © 2012 Springer Science+Business Media, LLC.

DOI： 10.1007/s10904-012-9725-5

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Language：English   Publisher：The Physical Society of Japan  

DOI： 10.1143/JPSJS.81SA.SA002

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This review presents highlights of our latest results of studies directed at developing protein-based smart nanotubes for biomedical applications. These practical biocylinders were prepared using an alternate layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into a nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. The obtained (PLA/HSA)(3) nanotubes (NTs) can be dispersed in aqueous medium and are hydrated significantly. Several ligands for HSA, such as zinc(II) protoporphyrin IX (ZnPP), were bound to the HSA component in the cylindrical wall. Similar NTs comprising recombinant HSA mutant, which has a strong binding affinity for ZnPP, captured the ligand more tightly. The Fe3O4-coated NTs can be collected easily by exposure to a magnetic field. The hybrid NTs bearing a single avidin layer as an internal wall captured biotin-labeled nanoparticles into the central channel when their particle size is sufficiently small to enter the pores. The NTs with an antibody surface interior entrapped human hepatitis B virus with size selectivity. It is noteworthy that the infectious Dane particles were encapsulated completely into the hollows. Other HSA-based NTs having an alpha-glucosidase inner wall hydrolysed a glucopyranoside to yield alpha-D-glucose. A perspective of the practical use of the protein-based NTs is also described.

DOI： 10.1039/c1nr11224d

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We describe template-assisted synthesis of protein nanotube arrays immobilized on solid substrates (e.g., glass plate) using a nanoporous polycarbonate membrane and the arrays' molecule-trapping capabilities in aqueous medium. The uniform array of human serum albumin-based nanotubes bearing an avidin surface interior captured efficiently fluorescein-labeled biotin in water.

DOI： 10.1246/cl.2011.1338

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This communication describes the synthesis and structure of multilayered protein nanotubes bearing a magnetite (Fe(3)O(4)) surface exterior, and their binding capability for zinc(II)-protoporphyrin IX (ZnPP) in aqueous medium. The nanotubes were fabricated using an alternating layer-by-layer (LbL) assembly of human serum albumin (HSA) and poly-L-arginine (PLA) in a track-etched polycarbonate (PC) membrane (pore size, 400 nm), which had been precoated in advance with Fe(3)O(4) nanoparticles. Dissolution of the PC template yielded Fe(3)O(4)(PLA/HSA)(3) nanotubes. SEM measurements revealed the formation of uniform hollow cylinders with 417 +/- 16 nm outer diameter and 56 +/- 7 nm wall thickness. TEM observations confirmed the homogeneous outer surface of Fe(3)O(4). In an aqueous medium, the nanotubes captured ZnPP into the swollen wall. The ZnPP-loaded protein nanotubes were collected by exposure to a magnetic field. Copyright (C) 2011 John Wiley & Sons, Ltd.

DOI： 10.1002/pat.1921

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Infectious hepatitis B virus (HBV), namely Dane particles (DP&apos;s), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 +/- 16 nm outer diameter and 59 +/- 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 x 10(7) M(-1) Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

DOI： 10.1021/ja1096122

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This report describes the synthesis and enzyme activities of multilayered protein nanotubes with an alpha-glucosidase (alpha GluD) interior surface. The nanotubes were prepared by using an alternating layer-by-layer (LbL) assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a track-etched polycarbonate (PC) membrane (pore size = 400 nm) followed by addition of aGluD as the last layer of the wall. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/alpha GluD nanotubes. SEM measurements revealed the formation of uniform hollow cylinders with (413 +/- 17) nm outer diameter and (52 +/- 3) nm wall thickness. In aqueous media, the nanotubes captured a fluorogenic glucopyranoside, 4-methyl-umbelliferyl-alpha-D-glucopyranoside (MUGlc), into their one-dimensional pore space and hydrolyzed the substrate efficiently to form alpha-D-glucose. We determined the enzyme parameters (Michaelis constant, K(M), and catalytic constant, k(cat) values) of the protein nanotubes. The several-micrometers-long cylinders were of sufficient length to be spun down by centrifugation at 4000 g, so the product could therefore be easily separated. Similar biocatalysts were prepared by complexation of biotinylated-alpha GluD into HSA-based nanotubes bearing a single avidin layer as an internal surface. The obtained hybrid nanotubes also exhibited the same enzyme activity for the MUGlc hydrolysis.

DOI： 10.1002/chem.201001937

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

Human serum albumin (HSA) is used clinically as a plasma expander in patients with hypoalbuminemia and can also function as a drug carrier. However, the administered HSA is readily eliminated from the blood circulation under pathological conditions, especially the nephrotic syndrome. In this study, we present data on the pharmacokinetics of a structurally defined HSA dimer [two HSA molecules that are cross-linked by reaction with 1,6-bis(maleimido) hexane via Cys34] in nephrotic rats and its superior circulation persistence, owing to the molecular size effect. The half-time (t(1/2)) of the HSA dimer persisted in the circulation 1.3 times longer than that of monomeric HSA in normal rats, primarily because of the suppression of the accumulation of the HSA dimer in the skin and muscle. In nephrotic rats, the t(1/2) of the HSA monomer decreased considerably, whereas the HSA dimer remained unaltered in the blood stream, similar to that for normal rats. As a result, the t(1/2) of the HSA dimer was 2-fold longer than that of the HSA monomer. This longer t(1/2) can be attributed to the fact that accumulation in the kidney and urinary excretion of the HSA dimer were significantly suppressed. The cross-linked HSA dimer shows a longer blood circulation than native HSA monomer in nephrotic rats, which can be attributed to the suppression of renal filtration and leakage into the extravascular space. This HSA dimer has the potential for use as a drug carrier, new plasma expander, and an artificial albumin-based oxygen carrier under a high glomerular permeability condition such as nephrosis.

DOI： 10.1124/dmd.109.031989

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A pair of myoglobins containing inherently distorted alpha-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared, and the functional consequence of intrinsic heme deformation was investigated. The visible absorption peaks of the myoglobin bearing the distorted heme exhibited a bathochromic shift, indicating that the heme was deformed in the protein pocket. Ligand affinities for the ferric myoglobin with the distorted heme were found to be higher than those of the myoglobin bearing the undistorted heme. The observation suggested that the iron atom was more displaced toward the proximal histidine to weaken the coordination of the water molecule. In the paramagnetic proton NMR spectrum of ferrous deoxy protein, the deformed heme caused a 3.2 ppm lower-field shift of the proximal histidine signal, supporting an enhanced iron-histidine interaction. The deformed heme in ferrous myoglobin lowered the oxygen and carbon monoxide affinities by 25- and 480-fold, respectively, and caused the cleavage of the iron-histidine bond in a fractional population of the nitric oxide derivative. These results demonstrate a distinctive controlling mechanism for ligand binding by the deformed heme. Upon the heme distortion, the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

DOI： 10.1021/bi1003553

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Solid nanotubes comprising alpha-Fe2O3 nanoparticles were prepared from iron-storage protein ferritin. Their structure, magnetic properties, and photocatalytic activities were characterized. The initial ferritin nanotube precursors were fabricated using alternating layer-by-layer depositions of poly-L-arginine (PLA) and ferritin into a track-etched polycarbonate membrane (pore diameter, 400 nm) with subsequent dissolution of the template. The obtained uniform cylinders of (PLA/ferritin)(3) (outer diameter, 410 +/- 14 nm) were calcinated at 500 degrees C under air, yielding reddish-brown iron oxide nanotubes. The one-dimensional hollow structure remained perfect, but its diameter, wall thickness, and maximum length were markedly diminished. Disappearance of the protein shell and the PLA layers were confirmed using IR and EDX spectroscopy. Subsequent SEM, TEM, and XPS measurements showed that the tubular walls comprise fine alpha-Fe2O3 nanoparticles with a 5 nm diameter. These of Fe2O3 nanotubes demonstrated superparamagnetic properties with a blocking temperature of 37 K and efficient photocatalytic activity for degradation of 4-chlorophenol.

DOI： 10.1021/nn901879d

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We describe molecular capturing properties of protein nanotubes with a controllable ligand binding affinity and size selectivity. These practical biocylinders were prepared using an alternating layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into the nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers Of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. Use of high molecular weight PLA (M(w) = ca. 70 000) yielded robust nanotubes, which are available as lyophilized powder. The (PLA/HSA)(3) nanotubes swelled considerably-in water, although the outer diameter was almost unaltered. Uranyl ion, 3,3&apos;-diethylthiacarbocyanine iodide, and-zinc(II) protoporphyrin IX (ZnPP) were bound to the HSA component in the cylinder wall. Similar nanotubes; comprising recombinant HSA mutant [rHSA(His)], which has a strong binding affinity for ZnPP, captured this ligand more tightly. Furthermore, addition of excess myristic acid released ZnPP from the tubes through a ligand replacement reaction. The hybrid nanotubes bearing a single avidin layer as an internal surface captured FITC-biotin efficiently. Biotin-labeled nanoparticles are also incorporated into the tubes when their particle size is sufficiently small to enter the pores. Subsequent TEM observation revealed a line of loaded nanoparticles (100 nm) in the one-dimensional space interior.

DOI： 10.1021/nn901474y

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Language：Japanese  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Complexation of iron(II) protoporphyrin IX (Fe2+ PP) into a genetically engineered heme pocket on recombinant human serum albumin (rHSA) creates an artificial hemoprotein which can bind O-2 reversibly at room temperature. Here we highlight a crucial role of a basic amino acid triad the entrance of the heme pocket in rHSA (Arg-114, His-146, Lys-190) for O-2 and CO binding to the prosthetic Fe2+ PP group. Replacing His-146 and/or Lys-190 with Arg resolved the structured heterogeneity of the possible two complexing modes of the porphyrin and afforded a single O-2 and CO binding affinity. Resonance Raman spectra show only one geometry of the axial His coordination to the central ferrous ion of the Fe2+ PP.

DOI： 10.1039/b909794e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Hemoglobin (fib, Mw: 64 500) and albumin (Mw: 66 500) are major protein components in Our circulatory system. On the basis of bioconjugate chemistry of these proteins, we have synthesized artificial O(2) carriers of two types, which will be useful as transfusion alternatives in clinical situations. Along with sufficient O(2) transporting capability, they show no pathogen, no blood type antigen, biocompatibility, stability, capability for long-term storage, and prompt degradation in vivo. Herein, we present the latest results, from our research on these artificial O(2) carriers, Hb-vesicles (HbV) and albumin-hemes. (i) HbV is a cellular type Hb-based O(2) carrier. Phospholipid vesicles (liposomes, 250 nm diameter) encapsulate highly purified and concentrated human Hb (35 g/dL) to mimic the red blood cell (RBC) structure and eliminate side effects of molecular Hb such as vasoconstriction. The particle surface is modified with PEG-cojugated phospholipids. thereby improving blood compatibility and dispersion stability. Manipulation of physicochemical parameters of HbV, such as O(2) binding affinity and suspension rheology, supports the use of HbV for versatile medical applications. (ii) Human serum albumin (HSA) incorporates synthetic Fe(2+)porphyrin (FeP) to yield unique albumin-based O(2) carriers. Changing the chemical structure of incorporated FeP Controls O(2) binding parameters. In fact. PEG-modified HSA-FeP showed good blood cumpatibility and O(2) transport in vivo. Furthermore, the genetically engineered heme pocket in HSA can confer O(2) binding ability to the incorporated natural Fe(2+)protoporphyrin IX (heme). The O(2) binding affinity of the recombinant HSA (rHSA)-heme is adjusted to a similar value to that of RBC through optimization of the amino acid residues around the coordinated O(2).

DOI： 10.1021/bc800431d

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAP SOC STUDY XENOBIOTICS  

Human serum albumin (HSA) is a versatile protein found at high concentration in blood plasma and binds a range of insoluble endogenous and exogenous compounds. We have shown that complexation of functional molecules into HSA creates unique proteins never seen in nature. Complexing an iron-protoporphyrin IX into a genetically engineered heme pocket of recombinant HSA (rHSA) generates an artificial hemoprotein, which binds O(2) reversibly in much the same way as hemoglobin. A pair of site-specific mutations, (i) introduction of a proximal histidine at the Ile-142 position and (ii) substitution of Tyr-161 with Phe or Leu, allows the heme to bind O(2). Additional modification on the distal side of the heme pocket provides rHSA(triple mutant)-heme complexes with a variety Of O(2) binding affinity. Complexing a carboxy-C(60)-fullerene (CF) into HSA generates a protein photosensitizer for photodynamic cancer therapy. Energy transfer occurs from a photoexcited triplet-state of HSA-CF (HSA-(3)CF*) to O(2), forming singlet oxygen ((1)O(2)). This protein does not show dark cytotoxicity, but induceds cell death under visible light irradiation.

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

The O-2 binding properties of complexes of iron(II) protoporphyrin IX with quadruple mutants of recombinant human serum albumin (rHSA) that provide axial His-186 coordination have been characterized; their O-2 binding parameters were similar to those of analogues having proximal His-185 and of human red blood cells.

DOI： 10.1246/cl.2009.776

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We present structure, photoexcited triplet-state property, and singlet oxygen (O-1(2)) generation capability of human serum albumin complexed with a tris(dicarboxymethylene)[60]fullerene C-3-isomer (HSA-CF). Small angle X-ray scattering measurements showed that a globular size-dimension and surface charge distribution of HSA were unaltered by monomolecular complexation of CF into the hydrophobic cavity. Laser flash photolysis to the HSA-CF solution yielded a photoexcited triplet state of the CF chromophore ((CF)-C-3*) with lifetime of 46 mu s. In the presence of O-2, energy transfer occurred from HSA -(CF)-C-3* to O-2 to generate O-1(2); the quenching rate constant [k(q)(O-2)] was determined to be 2.2 x 10(8) M-1 s(-1). The HSA-CF showed strong photoresistence relative to HSA complexed with a protoporphyrin IX. The quantum yield of O-1(2) production for this artificial protein was compared to those of other photosensitizing agent. The HSA-CF did not show a dark cytotoxicity, but induced cell death efficiently under visible light irradiation.

DOI： 10.1021/bc800207j

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We present the synthesis and Structure of various protein nanotubes comprised of an alternate layer-by-layer (LbL) assembly using a polycation as an electrostatic glue. The nanotubes were fabricated by sequential LbL depositions of positively charged polycations and negatively charged proteins into a porous polycarbonate (PC) membrane, followed by release of the cylindrical core by quick dissolution of the template with CH(2)Cl(2). This procedure provides a variety of protein nanotubes without interlayer cross-linking. The three-cycle depositions of poly-L-arginine (PLA) and human serum albumin (HSA, M(w)=66.5 kDa) into the porous PC template (pore diameter, D(P)=400 nm) yielded well-defined (PLA/HSA)(3) nanotubes with all outer diameter of 419 +/- 29 nm and a wall thickness of 46 +/- 8 nm, revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. The outer diameter of the tubules can be controlled by the pore size of the template (200-800 nm), whereas the wall thickness is always constant, independent of the D(P) value. The (PEI/HSA)(3) (PEI: polyethylenimine) nanotubes showed a slightly thin wall of 39 5 nm. CD spectra of the multilayered (PEI/HSA)(n), film on a flat quartz plate suggested that the secondary structure of HSA between the polycations was almost the same as that in aqueous solution. The three-cycle LbL depositions of PLA and ferritin (M(w) = 460 kDa) or myoglobin (Mb, M(w) =1.7 kDa) into the porous PC membrane also gave cylindrical hollow structures. The wall thickness of the (PLA/ferritin)(3) and (PLA/Mb)(3) nanotubes were 55 +/- 5 nm and 31 +/- 4 nm; it depends oil the globular size of the protein (ferritin &gt; HSA &gt; Mb). The individual ferritin molecule was clearly seen in the tubular walls by SEM and TEM measurements.

DOI： 10.1002/chem.200800771

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The iron complex of oxypyriporphyrin, a porphyrinoid containing a keto-substituted pyridine, was coupled with apomyoglobin. The reconstituted ferric myoglobin was found to be five-coordinate without iron-bound water molecules. The anionic ligands such as CN(-) and N(3)(-) bound the myoglobin with high affinities, while neutral imidazole did not. The IR observation indicated that the azide complex was pure high-spin, although the corresponding native protein was in the spin-state equilibrium. The reduced myoglobin was five-coordinate but exhibited no measurable affinity for O(2). The affinity for CO was lowered down to 1/2400 as compared with native myoglobin. These anomalies were ascribed to the deformation in the iron coordination core after the replacement of one of the four pyrroles with a larger pyridine ring. The ligand binding analyses for the ferric and ferrous myoglobin suggest that the proximal histidine pulls the iron atom from the deformed core to reduce the interaction between the iron and exogenous ligands. Similarity of the reconstituted myoglobin with guanylate cyclase, a NO-responsive signaling hemoprotein, was pointed out.

DOI： 10.1021/ic801406x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Cylindrical protein nanotubes comprising a layer-by-layer (LbL) assembly of human serum albumin (HSA) with polycations [polyallylamine hydrochloride (PAH), poly-L-arginine hydrochloride (PLA), and poly-L-lysine hydrobromide (PLL)] have been prepared using template synthesis with a porous anodic aluminum oxide (AAO) membrane. Visible absorption spectra of the multilayered thin film of the polycation and HSA on a quartz plate showed alternate LBL film formation of (polycation/HSA)(n).

DOI： 10.1246/cl.2008.972

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-V C H VERLAG GMBH  

We present the O-2 binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(II) protoporphyrin IX as a prosthetic heme group. Iron(III) protoporphyrin IX (hemin) is bound within subdomain IB of HSA with weak axial coordination by Tyr-161. In order to confer O-2 binding capability to this naturally occurring hemoprotein: (i) a proximal histidine was introduced into position Ile-142; and (ii) the coordinated Tyr-161 was replaced with hydrophobic Leu using site-directed mutagenesis. it provided a recombinant HSA double-mutant [rHSA(l142H/ Y161L)=rHSA(HL)]. The rHSA(HL)-heme formed a ferrous five-coordinate high-spin complex with axial ligation of His-142 under an Ar atmosphere. This artificial hemoprotein binds O-2 at room temperature. Laser flash photolysis experiments demonstrated that O-2 rebinidng to rHSA(HL)-heme displays monophasic kinetics, whereas the CO recombination process obeyed a double-exponential pattern. This might be attributable to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O-2 binding affinity of rHSA(HL)-heme was considerably lower than those of R-state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O-2 dissociation rate constant. The third mutations have been introduced into the distal side of the heme (at position Leu-185 or Arg-186) to increase the O-2 binidng affinity. The rHSA(HL/L185N)-heme showed high O-2 binding affinity (P-1/2(O2) : 1 Torr), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and which is rather close to those of HID (R-state) and Mb. Furthermore, replacement of polar Arg-186 with Leu or Phe adjusted the O-2 binding affinity (P-1/2(O2)) to 10 Torr, which is almost equivalent to value for human red blood cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Tumor-cell hypoxia is one of the main factors inducing radioresistance. Enhanced tumor oxygenation has previously been achieved in an animal model using the synthetic heme-based oxygen carrier &apos;albumin-heme&apos; (recombinant human serum albumin-Fe cyclohexanoil heme; rHSA-FeP). The present study was done to determine whether rHSA-FeP enhances the radiation response in an experimental tumor model. Male Donryu rats and LY80, a variant of the syngenic liver ascites tumor, were used. A total of 1 x 10(6) cells were injected into the subfascial tissue of the right thigh. The rats were divided randomly into five groups: sham (tumor implantation and sham operation); rHSA-FeP; irradiation; rHSA + irradiation; and rHSA-FeP + irradiation. Six days after, under general anesthesia, intra-arterial administration of 10 mL/kg of either 5% rHSA solution or oxygenated rHSA-FeP solution at 2.5 mL/min was done and a dose of 20 Gy was given. There were significant differences in tumor growth between the sham and irradiation groups, and between the sham and rHSA-FeP + irradiation groups. Tumor growth delay was observed and differences were significant between the sham and irradiation groups, and between the irradiation and rHSA-FeP + irradiation groups. In the present study, rHSA-FeP itself had a slight effect on tumor growth without irradiation. Enhancing the effect of rHSA-FeP on the radiation response is responsible in part for the oxygen-carrying property of rHSA-FeP. In conclusion, rHSA-FeP is a candidate radiation-enhancing drug. Arterial infusion of rHSA-FeP may serve as a local oxygenation method that enhances the radiation effect.

DOI： 10.1111/j.1349-7006.2008.00811.x

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We describe the significant difference in the O-2 binding affinities of human serum albumin (HSA) incorporating 5,10,15,20-tetrakisf(alpha,alpha,alpha,alpha-o-(1'-methylcyclohexanamido)phenyl}porphinatoiron(II) with a covalently linked 1-methyl-L-histidine or 3-methyl-L-histidine [HSA-FeP(I-MHis), HSA-FeP(3-MHis)]. The HSA-FeP(3-MHis) showed an extraordinarily high O-2 binding affinity (P-1/2 = 0.2 Torr, 25 degrees C, pH 7.4), which is close to those of relaxed-state hemoglobin and myoglobin. However, replacement of the 3-methyl-L-histidine moiety in FeP(3MHis) by 1-methyl-L-histidine caused a 35-fold reduction in O-2 affinity; the P-1/2 value of HSA-FeP(I-MHis) (22 Tort, 37 degrees C, pH 7.4) is almost identical to that of human red blood cells. Results of kinetic studies indicate that the low O-2 binding affinity of FeP(l -MHis) is, O-2 predominantly manifested in the high O-2 dissociation rate constant. In a toluene solution, an identical relationship in the O-2 binding property was similarly observed for FeP(1-MHis) and FeP(3 MHis). The axial Fe-N(1-MHis) coordination might be restrained by steric interaction between the 4-methylene group of the histidine and the porphyrin plane.

DOI： 10.1021/bc700400n

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O-2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG(2)) or 5-kDa (PEG(5))] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG(2)(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 mu m thickness), and (ii) a bottom layer of an amorphous phase (5 mu m thickness). The condensed solution provided a thick membrane (70 mu m), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O-2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O-2-binding affinity of the PEG(2)(HSA-FeP) membrane (P-1/2 = 40 Tort, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O-2 as well. These O-2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.

DOI： 10.1021/bc070086m

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O-2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O-2 reversibly: (i) IIe-142 -&gt; His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161 -&gt; Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O-2 binding equilibrium. The coordination structures and ligand (O-2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O-2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O-2. In marked contrast, HSA(HL/L185N)-heme showed very high O-2 binding affinity (P-1/2(2)o 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O-2 binding primarily by reducing the O-2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O-2 binding affinity (P-1/2(O2) 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O-2 transport.

DOI： 10.1021/ja074179q

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Artificial hemoprotein nanotubes have been prepared by a layer-by-layer deposition technique with human serum albumin (HSA) incorporating the synthetic heme (FeP) [HSA-FeP] using an anodic porous alumina template; each of the liberated tubules has a very uniform outer/inner diameter and can reversibly bind dioxygen (O-2) at 25 degrees C.

DOI： 10.1039/b704135g

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Recombinant human serum albumin (HSA) including the synthetic iron(II)-porphyrin (FeP), albumin-heme (HSA-FeP), is a unique O-2-carrying plasma hemoprotein as a red blood cell substitute. We have investigated the possible influence of HSA-FeP on the complement system and platelet activation in vitro. The amounts of the serum complement titer CH50 and terminal complement complex SC5b-9 of human blood serum, incubated with HSA-FeP (10, 20, and 40 vol %), were almost the same as those of the corresponding samples with HSA. The effect of HSA-FeP on the platelet reactivity has been demonstrated by conformational changes in the membrane glycoprotein IIb/IIIa and surface expression of an a-granule membrane protein P-selectin. Platelet activation in response to the ADP-stimulation was not influenced by the presence of HSA-FeP. it can be concluded that the albumin-heme solution does not facilitate the immunological reaction and platelet activation. Moreover, a 20% exchange transfusion with HSA-FeP into anesthetized rats has been performed to evaluate the circulation and blood parameters for 6 h. Time course changes in all parameters showed features identical to the control group (without infusion) and FISA group. (c) 2007 Wiley Periodicals, Inc. J Biomed MaterRes 81A: 82-1-826, 2007.

DOI： 10.1002/jbm.a.31016

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

meso-Tetrakis(alpha,alpha,alpha,alpha-o-(1-methylcyclohexanamido)phenyl)porphinatoiron (III) bearing a proximal histidyl group at the beta-pyrrolic position via an acyl bond (4c) has been synthesized. Human serum albumin (HSA) incorporating the ferrous complex (4d) formed a stable O-2 adduct under physiological conditions (pH 7.4, 37 degrees C). Although an electron-withdrawing acyl group is attached to the porphyrin periphery, the O-2-binding affinity of HSA-4d was slightly higher than that of a similar analogue with a histidyl-alkylene group (2d).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMERICAN PHYSICAL SOC  

Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

DOI： 10.1103/PhysRevLett.98.208101

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)-protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H-2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt)] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA( His)], by a His-142 coordination. Both the rHSA(wt)-ZnPP and rHSA( His)-ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (tau(T)) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA-(ZnPP)-Zn-3* by MV2+. The quenching rate constant (k(b)) and backward electron transfer rate constant (k(b)) were determined to be 1.4 x 10(7) and 4.7 x 10(8) M-1 s(-1) for rHSA(wt)-ZnPP. In the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H-2 takes place. The efficiency of the photoproduction of H-2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl) porphinatozinc(II) (ZnTMPyP4+), under the same conditions.

DOI： 10.1021/ja0656806

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Poly(ethylene glycol) (PEG) conjugated recombinant human serum albumin (HSA) incorporating the synthetic iron-porphyrin (FeP) [PEGylated albumin-heme, PEG(HSA-FeP)] is a unique albumin-based oxygen carrier as a red blood cell (RBC) substitute. The physiological responses to an exchange transfusion with PEG(HSA-FeP) into an acute anemia rat model were investigated. After a 65% isovolemic hemodilution with HSA, a 30% volume of the circulatory blood was withdrawn, affording a hemorrhaged state. The circulation parameters, blood parameters, renal cortical oxygen partial pressure [PtO2(R)], and muscle tissue oxygen partial pressure [PtO2(M)] were continuously monitored. The intravenous infusion of PEG(HSA-FeP) restored the reduced levels of the mean arterial pressure, heart rate, respiration rate, mixed venous PO2, and arterial PCO2. The increased arterial PO2 and pH also returned to their basal values. These effects were almost to the same extent as those observed after the administration of the RBC suspension. The relatively low recovery in PtO2(R) and PtO2(M) might be due to the Langirmir-type oxygen binding profile of PEG(HSA-FeP) (Hill coefficient: 1.0). All the animals survived during the experiments. In contrast, those injected with HSA died within 41 min. The PEG(HSA-FeP) solution is an oxygen-carrying plasma expander which can be used as a resuscitative fluid for hemorrhagic shock. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2006.04.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Artificial O-2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)-porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene alycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the theological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O-2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O-2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O-2-carrying plasma expander as a red cell substitute.

DOI： 10.1021/bc050315+

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We have found that recombinant human serum albumin (HSA) incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer can reversibly bind and release O-2 under physiological conditions (pH 7.3, 37 degrees C)like hemoglobin and myoglobin. beta-2-Methylimidazolyl-tailed porphyrinatoirons (6a, 6b) are synthesized via four steps from the atropisomers of tetrakis(o-aminophenyl)porphyrin. The stereochemistry of the a,(X,(X,P-conformer has been determined by NMR spectroscopy. 6a and 6b form stable O-2-adduct complexes in toluene solution at room temperature. The association rate constants of O-2 are 3.1- and 1.9-fold lower than those of the corresponding alpha,alpha,alpha,alpha-conformers (1a, 1b), indicating that the three substituents (cyclohexanamide or pivalamide groups) are close to each other on the porphyrin platform and construct a narrow encumbrance around the O-2-coordination site. Although 6a and 6b are incorporated into the hydrophobic domains of HSA to produce the albumin-heme hybrid, only HSA-6a can bind 02 in aqueous medium. The cyclohexanamide fences are necessary for the tailed porphyrinatoiron to form a stable O-2-adduct complex under physiological conditions. The O-2-binding affinity (P-1/2) of HSA-6a is 45 Torr (37 degrees C), and the O-2 transporting efficiency between lungs and muscle tissues in the human body is estimated to be identical to that of human red blood cells. The HSA-6a solution will become one of the most promising materials for red blood cell substitutes, which can be manufactured on an industrial scale.

DOI： 10.1021/bc050154+

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Language：Japanese   Publishing type：Research paper (international conference proceedings)  

We have evaluated the dioxygen binding properties of albumin hybrid incorporating porphyrinatoirons having histidine derivative [FeP(His), FeP(MHis)]. They were synthesized via 9 steps from 5,10,15,20-tetrakis(o- aminophenyl)porphyrin. FeP(His) binds histidine and FeP(MHis) binds 1-methylhistidine as a proximal base. Both hemes could be incorporated into human serum albumin (HSA) providing HSA hybrid [HSA-FeP(His) or HSA-FeP(MHis)] in the buffer solution (pH 7.3). HSA-FeP(MHis) showed a low O 2-binding affinity (P50: 15 Torr) and a high O 2-dissociation rate constant in comparison to HSA-FeP(His). This is due to the steric hindrance for the coordination of the proximal base derived from the methylene group at 4-posititon of imidazole ring in FeP(MHis). HSA-FeP(MHis) can be utilized as a new artificial O2-therapeutics.

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Ltd  

Albumin-heme is a unique product-an oxygen transporter that is totally synthetic. By this approach, human serum albumin is produced in a recombinant yeast system, and then synthetic heme is coordinated to its surface. Up to 8 heme groups per molecule have been incorporated so far. Albumin-heme has been prepared to have a P50 similar to that of red blood cells, but the oxygen binding is not cooperative. It avidly binds NO, but is not hypertensive in preliminary animal tests. It appears to be as stable with regards to oxidation as native human hemoglobin. A number of early preclinical tests have been performed, including biocompatibility and effects on coagulation, and no significant toxic effects have been noted. While it is still early in the development of albumin-heme as a therapeutic agent for use in humans, and the cost of production is likely to be high. It is an intriguing product that could find use in specialized applications such as oxygenating tumors to increase the effects of radio- or chemotherapy. Results have shown O2-transporting efficacy and initial clinical safety of the recommended human serum albumin (rHSA)-heme solution that allows a patient to undergo further advanced preclinical testing of this synthetic O2 carrying plasma protein. Exchange transfusion with rHSA-heme into beagles is now under investigation. Furthermore, rHSA-heme as a monomolecular O2 carrier was tested for its ability to increase O2 tension in the hypoxia of the solid tumor rat model. © 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/B978-012759760-7/50057-3

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Language：Japanese   Publishing type：Research paper (international conference proceedings)  

Hemin is bound within a hydrophobic cavity in subdomain IB of human serum albumin (HSA). It has been shown that the introduction of a proximal His into the subdomain IB of HSA by site-directed mutagenesis allows a reversible O 2 binding of the prosthetic heme group. We have prepared new recombinant HSA (rHSA) having As, Gin or His at the 185 position as a distal-base and evaluated its effect on the O2-binding property. The introduction of 185-Asn provided an 18-fold larger O2-binding affinity compared to the mutant having 185-Leu. In contrast, 185-Gln and -His bound to the central iron to form a six-coordinate low-spin complex under argon and be oxidized by exposure of O2.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The binding properties of O-2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O-2. To confer O-2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/ Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O-2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O-2 binding to the heme. In contrast, the single mutant rHSA(I142H)heme could not bind O-2, suggesting that removal of Y161 is necessary to confer reversible O-2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)heme were biphasic, whereas O-2 rebinding exhibited monophasic kinetics. This could be due to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O-2 binding affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O-2 dissociation rates. Changing Leu161 to Phe-161 at the distal side increased the association rates of both O-2 and CO, which resulted in enhanced binding affinity.

DOI： 10.1021/ja054819u

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shelf albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau(1/2)) of the I-125-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O-2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O-2-binding properties (O-2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume Of O-2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.

DOI： 10.1021/bm050454u

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Synthetic iron(II) porphyrin (FeP) is equivalently incorporated into recombinant Thermotoga maritima xylanase B (TMX; family F/10 of glycoside hydrolase), producing a heat-resistant artificial hemoprotein (TMX-FeP) that can bind and release oxygen (O-2) in aqueous medium (pH 7.3, 25 &DEG; C) in the same manner as hemoglobin and myoglobin. The oxygenated species was sufficiently stable; the half-lifetime against the ferric state (&tau;(1/2)) was 5 h. This O-2-carrying hemoprotein showed a high degree of thermal stability over a wide range of temperatures up to 90 &DEG; C (&tau;(1/2) = 5 min at 90 &DEG; C and 9 min at 75 &DEG; C). Dictyoglomus thermophilum xylanase B (DTX; family G/11) also incorporates FeP, and DTX-FeP showed identical O-2-binding parameters and thermostability. TMX-FeP is capable of catalyzing the &beta;-1,4-D-xylan hydrolysis reaction. Its larger K-m value compared to that of TMX itself suggested competitive FeP binding to the active site of the host enzyme.

DOI： 10.1021/bm0492551

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

The Co-Na heterobinuclear polymer complex based on Salen Schiff base and crown ether have been prepared by a method of interfacial polymerization without the presence of a phase transfer catalyst. The catalytic behavior of the Co-Na heterobinuclear polymer complex in aerobic oxidation of cyclohexene has been studied.

DOI： 10.1081/MA-200046986

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley & Sons  

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Publishing type：Research paper (scientific journal)  

Since the discovery of a red-colored saline solution of a heme derivative that reversibly binds and releases oxygen (1983), significant efforts have been made to realize an oxygen infusion as a red cell substitute based on the sciences of both molecular assembling phenomena and macromolecular metal complexes. The authors have specified that hemoglobin (Hb)-vesicles (HbV) and recombinant human serum albumin-hemes (rHSA-heme) would be the best systems that meet the clinical requirements. (A) Hb is rigorously purified from outdated, donated red cells via pasteurization and ultrafiltration, to completely remove blood type antigen and pathogen. The HbV encapsulates thus purified concentrated Hb solution with a phospholipid bimolecular membrane (diameter, 250 nmø), and its solution properties can be adjusted comparable with blood. Surface modification of HbV with a water-soluble polymer ensures stable dispersion state and storage over a year at 20°C. In vivo tests have clarified the efficacy for extreme hemodilution and resuscitation from hemorrhagic shock, and safety in terms of biodistribution, metabolism in reticuloendothelial system (RES), clinical chemistry, blood coagulation, etc. The HbV does not induce vasoconstriction thus maintains blood flow and tissue oxygenation. (B) rHSA is now manufactured in Japan as a plasma-expander. The rHSA can incorporate eight heme derivatives (axial base substituted hemes) as oxygen binding sites, and the resulting rHSA-heme is a totally synthetic O2-carrier. Hb binds endothelium-derived relaxation factor, NO, and induces vasoconstriction. The rHSA-heme binds NO as Hb does, however, it does not induce vasoconstriction due to its low pI (4.8) and the resulting low permeability across the vascular wall (1/100 of Hb). A 5%-albumin solution possesses a physiologic oncotic pressure. Therefore, to increase the O2-transporting capacity, albumin dimer is effective. Albumin dimer can incorporate totally 16 hemes with a regulated oncotic pressure. The rHSA-heme is effective not only as a red cell substitute but also for oxygen therapeutics (e.g. oxygenation for tumor). Significant efforts have been made to produce HbV and rHSA-heme with a facility of Good Manufacturing Practice (GMP) standard, and to start preclinical and finally clinical trials. Copyright © 2005 John Wiley & Sons, Ltd.

DOI： 10.1002/pat.559

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Tetrakis{alpha,alpha,alpha,alpha-o-pivalamido)phenyl}porphinatoiron(II) with a bifunctional tail possessing an axially coordinated imidazolyl group and a protein attachable succinimidyl(glutamyl) group (FeP-GluSu) has been synthesized. It can efficiently react with the lysine residues of recombinant human serum albumin (rHSA), giving a new albumin-heme conjugate [rHSA(FeP-Glu)]. MALDI-TOFMS showed a distinct molecular ion peak at m/z 70 643, which indicates that three FeP-Glu molecules were covalently linked to the rHSA scaffold. The binding number of FeP-Glu is approximately three (mol/mol) and independent of the mixing ratio. The CD spectrum and Native PAGE revealed that the albumin structure remained unaltered after the covalent bonding of the hemes. This rHSA(FeP-Glu) conjugate can bind and release O-2 reversibly under physiological conditions (pH 7.3, 37 degreesC) in the same manner as hemoglobin and myoglobin. The O-2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O-2-binding affinity [P-1/2(O2): 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O-2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.

DOI： 10.1021/bc049859m

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Authorship：Lead author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG TOKYO  

Recombinant human serum albumin (rHSA) incorporating tetraphenylporphyrinatoiron(II) derivative with four pivaloylamino substituents (FepivP), albumin-heme, is an entirely synthetic hemoprotein that can reversibly bind and release O-2 under physiological conditions. We have recently found that replacing the substituent groups of FepivP with more hydrophobic 1-methylcyclohexanoylamino groups, affording FecycP, substantially stabilizes the formed O-2-adduct complex. The O-2- and CO-binding abilities and blood compatibility of this new rHSA-heme hybrid (rHSA-FecycP) have been investigated by spectroscopy. The maximum number of FecycP binding to one albumin was determined to be eight. Because the isoelectric point and circular dichroism (CD) spectral pattern were identical to those of rHSA itself, the two-dimensional structure of the host albumin could be unchanged after the incorporation of FecycP. Laser-flash photolysis experiments gave the association and dissociation rate constants for O-2 and CO (k(on), k(off)). The rebinding kinetics of these gaseous ligands consists of multiple exponentials. We conjectured that the O-2- and CO-binding reactions are affected by the molecular environment around each of the active heme sites. rHSA-FecycP showed almost the same O-2-binding affinity(P-1/2(O2) 34 torr at 37degreesC) and thermodynamic parameters (DeltaH, DeltaS) for the oxygenation as rHSA-FepivP. In contrast, the half-life of the O-2-adduct complex (9h, 37degreesC) became significantly longer than that of rHSA-FepivP (by a factor of 4.5), which is close to that of myoglobin. The obtained red solution was stable and demonstrated a long shelf life (&gt;2 years) at room temperature. The equivalent mixture of rHSA-FecycP and whole blood exhibited no coagulation or precipitation, indicating its high blood compatibility.

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Language：Japanese   Publishing type：Research paper (international conference proceedings)  

The binding properties of O2 and CO to artificial hemoproteins made of recombinant human serum albumin (rHSA) mutants with prosthetic heme group have been kinetically characterized. Five rHSA(mutant)s (rHSA(A): l142H, rHSA(B): l142H/Y161L, rHSA(C): l142H/Y161F, rHSA(D): l142H/ Y161L/L185N, rHSA(E): l142H/Y161F/L185N) were prepared. The all rHSA-heme except for rHSA(A)-heme formed an O2-adduct complex at room temperature. Laser flash photolysis experiments showed that the both O2 and CO binding processes to rHSA(mutant)-hemes were composed of two phases. It could be due to the two different geometries of the axial imidazole coordination arise from the two orientations of the porphyrin plane in the heme pocket. Changing Phe-161 to Leu-161 at the distal-side increased the association rate constants of O 2 and CO due to the increase of steric hindrance for binding them. Changing Leu-185 to Asn-185 at the distal-side decreased the dissociation rate constants of O2 due to the increase of polarity around the O 2-binding site.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Recombinant human serum albumin (rHSA) incorporating the synthetic heme "albumin-heme" is an oxygen-carrying plasma protein that has the potential to be a red blood cell substitute. The physiological responses to a 30% exchange transfusion with two types of albumin-heme (rHSA-FecycP, rHSA-FepivP) solutions after 70% isovolemic hemodilution with 5 g/dL rHSA were investigated using anesthetized rats. The circulation parameters, blood parameters, renal cortical oxygen pressure (pO(2)), and muscle tissue pO(2) were carefully monitored for 60 min after the injection. The declined mean arterial pressure and the mixed venous partial pO(2) signiicantly recovered to 70.8 and 91.9% of the basal values by intravenous infusion of albumin-hemes, respectively. The lowered renal cortical pO(2) also increased, indicating oxygen transport by this synthetic hemoprotein. The administration of albumin-heme into the acute anemia rat model after hemorrhage improved the circulatory volume and resuscitated the shock state. Both rHSA-FecycP and rHSA-FepivP transported oxygen through the body. (C) 2004 Wiley Periodicals, Inc.

DOI： 10.1002/jbm.a.30200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Recombinant human serum albumin (rHSA) incorporating synthetic heme with a covalently linked proximal base (albumin-heme [rHSA-heme]) is an artificial 0, carrier that can transport 0, like hemoglobin does in the blood stream. To evaluate the clinical safety of this compound, 20% and 40% exchange transfusions with rHSA-heme into anesthetized rats were followed by blood biochemical tests and histopathologic observations for 7 days. In the 20% rHSA-heme group, a total of 30 analytes by blood biochemical tests showed almost the same values as those observed in the reference 20% rHSA group. Although some abnormal values for liver parameters were found-in the 40% rHSA-heme group, they returned to normal after 7 days. Histopathologic observations indicated that the administration of rHSA-heme in a volume of 20% total blood volume did not produce any negative side effects on the vital organs.

DOI： 10.1097/01.MAT.0000144361.60280.DA

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pl: 4.8) and denaturing temperature (65degreesC). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the I-125-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis{(1-methylcyclohexanamido)phenyl}porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O-2 under physiological conditions (37degreesC, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O-2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O-2-recombination reaction. The O-2-binding affinity and the O-2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2004.08.010

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

DOI： 10.1021/ja046022t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Recombinant human serum albumin (rHSA) incorporating 2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-[tetrakis {alpha,alpha,alpha,alpha-0-(1-methylcyclohexanoyl)amino}-phenyllporphinatoiron(II) [albumin-heme (rHSA-heme)] is an artificial hemoprotein which has the capability to transport O-2 in vitro and in vivo. A 20% exchange transfusion with rHSA-heme into anesthetized rats has been performed to evaluate its clinical safety by monitoring the circulation parameters and blood parameters for 6 h after the infusion. Time course changes in all parameters essentially showed the same features as those of the control group (without infusion) and rHSA group (with administration of the same amount of rHSA). Blood biochemical tests of the withdrawn plasma at 6 h after the exchange transfusion have also been carried out. No significant difference was found between the rHSA-heme and rHSA groups, suggesting the initial clinical safety of this entirely synthetic O-2-carrier as a red-cell substitute. (C) 2004 Wiley Periodicals, Inc.

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Language：Japanese   Publisher：体液・代謝管理研究会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The simple one-pot reaction of protoporphyrin IX and omega-(N-imidazolyl) alkylamine or O-methyl-L-histidyl-glycine with benzotriazol-1-yl-oxytris(dimethylamino) phosphonium hexafluorophosphate at room temperature produced a series of protoporphyrin IX species with a covalently linked proximal base at the propionate side-chain. The central iron was inserted by the general FeCl2 method, converting the free-base porphyrins to the corresponding protoheme IX derivatives. Mesoporphyrin IX and diacetyldeuteroporphyrin IX analogues were also prepared by the same procedure. The Fe( II) complexes formed dioxygen (O-2) adducts in dimethylformamide at 25degreesC. Some of them were incorporated into the hydrophobic domain of recombinant human serum albumin (rHSA), providing albumin-heme hybrids (rHSA-heme), which can bind and release O-2 in aqueous media (pH 7.3, 25degreesC). The oxidation process of converting the dioxygenated heme in rHSA to the inactive Fe(III) state obeyed first-order kinetics, indicating that the l-oxo dimer formation was prevented by the immobilization of heme in the albumin scaffold. The rHSA-heme, in which the histidylglycil tail coordinates to the Fe(II) center, showed the most stable O-2 adduct complexes.

DOI： 10.1039/b409017a

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Recombinant human serum albumin complexed with tetraphenylporphinatoiron(II) derivative, "albumin-heme (rHSA-FeP)". is a synthetic oxygen (O-2)-carrying plasma hemoprotein, which becomes a new class of red blood cell substitute. The UV-vis. absorption and ESR spectroscopy revealed that rHSA-FeP formed six-coordinate nitrosyl complex after exposure of nitric oxide (NO) gas. Although the NO-binding affinity of rHSA-FeP (p(1/2)(NO): 1.7 x 10(-6) Torr, pH 7.3, 25 degreesC) is 9-fold higher compared to that of hemoglobin (Hb), the administration of this artificial hemoprotein solution into anesthetized rat does not induce an acute increase in blood pressure (hypertension), which is often observed in Hb-based O-2-carriers due to the depletion of NO (endothelial derived relaxing factor).

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Novel molecular energy and electron transfer assemblies in vesicular form, which are made of self-organized amphiphilic porphyrins bearing phospholipid-like substituents (lipid-porphyrins), have been photochemically characterized. Tetraphenylporphyrin (TPP) derivatives with four dialkylphosphocholine groups [free-base (1 a), Zn2+ complex (1b), and Fe3+ complex (1c)] are spontaneously associated in water to form spherical unilamellar vesicles with a diameter of 100-150 nm. Exciton calculations based on the bilayered sheet model of 1b, which has a porphyrin packing similar to that seen in the triclinic unit cell of the Zn2+TPP crystals, reproduced the Soret band bathochromic shift appearing in the aqueous solution of 1b well. The UV/Vis absorption spectrum of the 1a/1b hybrid vesicles (molar ratio: 1/1) showed no electronic interaction between the two porphyrin chromophores in the ground state, but efficient intermolecular singlet-singlet energy transfer took place from the excited 1b donors to the I a acceptor within the vesicle. Near-field scanning optical microspectroscopy of the 1a/1b vesicles on a graphite surface also showed only free-base porphyrin fluorescence. The efficiency of the energy transfer was 0.81 and the rate constant was 3.1 x 10(9) s(-1). On the other hand, protoporphyrin IX bearing two alkylphosphocholine propionates (2) was incorporated into the la or 1c bilayer vesicles (ca. 100 nm phi, molar ratio: 1a/2 or 1c/2=10). The UV/Vis absorption spectrum showed that 2 was successfully anchored into the fluid alkylene region of the membrane without stacking. Photoirradiation (lambda(ex): 390 nm) of the 1c/2 vesicles in the presence of triethanolamine led a vectorial electron transfer from the outer aqueous phase to the membrane center, which allowed reduction of the ferric ion of the Fe3+TPP platform.

DOI： 10.1002/chem.200305013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

Recombinant human serum albumin (rHSA) incorporating the iron(II) complex of the tetraphenylporphyrin derivative (FepivP or FecycP) is a synthetic O-2-carrying hemoprotein [albumin-heme (rHSA-FepivP or rHSAF-FecycP)], which acts as a red blood cell substitute. The association and dissociation behavior of FepivP and FecycP with rHSA has been initially investigated by isothermal titration calorimetry. A strong heat release appeared after the injection of albumin-heme into a large molar excess of rHSA. This exothermic enthalpy change was due to the transference of hemes to the other free albumins. The difference in the heme binding affinity to rHSA can be manifested in the enthalpy term. Copyright (C) 2003 John Wiley Sons, Ltd.

DOI： 10.1002/pat.418

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Recombinant human serum albumin including 2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivaloylamino)phenylporphinatoiron(II) (albumin-heme; rHSA-FeP) is a synthetic hemoprotein that has sufficient capability to reversibly bind and release O-2 under physiological conditions (pH 7.3, 37degreesC) similar to hemoglobin and myoglobin. In order to use this albumin-based O-2 carrier as a new class of red blood cell substitutes, its compatibility with blood cell components carefully was investigated in vitro. After the addition of the rHSA-FeP solution into whole blood at 10, 20, and 44 vol%, the FeP concentration in the plasma phase remained constant for 6 h at 37degreesC in each group, and no significant time dependence was observed in the numbers of red blood cells, white blood cells, or platelets. The microscopic observations clearly showed that the shapes of the red blood cells had not been deformed during the measurement period. With respect to the blood coagulation parameters (prothrombin time and activated partial thromboplastin time), the coexistence of rHSA-FeP had only a negligibly small influence. Also the blood compatibility under dynamic flow conditions was evaluated using a microchannel array flow analyzer. All these results suggest that the albumin-heme has no effect on the morphology of blood cell components in vitro. (C) 2003 Wiley Periodicals, Inc.

DOI： 10.1002/jbm.a.10573

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central  

Background: Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex. Results: We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes
this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid
the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161. Conclusion: The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties.

DOI： 10.1186/1472-6807-3-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Human serum albumin incorporating the iron(II) complex of protoporphyrin IX having an axially coordinated histidylglycyl-propionate formed a. dioxygen-adduct complex in aqueous media (pH 7.3). The O-2-binding affinity (P-1/2) was 0.1Torr at 25degreesC.

DOI： 10.1246/cl.2003.504

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The administration of extracellular, hemoglobin-based oxygen carriers often elicits an acute increase in blood pressure by vasoconstriction. This side effect is now recognized to be due to the depletion of nitric oxide (endothelial-derived relaxing factor) by the extravasuated hemoglobins. We have recently found that the administration of a recombinant human serum albumin (rHSA)-based oxygen carrier involving synthetic tetraphenyporphinatoiron(II) derivative (FeP) (rHSA-FeP) does not induce such hypertensive action, because of its low permeability through the vascular endothelium. The heart rate responses after the rHSA-FeP injection were also negligibly small. Visualization of the intestinal microcirculatory changes clearly revealed the widths of the venule and arteriole to be fairly constant. The entirely synthetic rHSA-FeP becomes a promising material as a new type of red blood cell substitute. (C) 2002 Wiley Periodicals Inc.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

alpha-Cyclodextrin (alphaCD)-penetrating 2-methyl-1-phenethylimidazole coordinates to the zinc(II) and iron(II) complexes of meso-tetrakis[o-(N-methyl) pyridinium] porphyrinate, giving non-covalently linked alphaCD-porphyrin ensembles; the iron(II) complex can reversibly bind and release dioxygen in aqueous DMF solution.

DOI： 10.1039/b208882g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

meso-Tetrakis(alpha, alpha, alpha, alpha-o-pivalamidophenyl)porphinatoiron(II) derivative is incorporated into thermophilic xylanases, giving novel thermostable synthetic hemoproteins, which can form a dioxygen adduct in aqueous media (pH 7.3) at 90 degreesC.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

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Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

An amphiphilic tetraphenylporphyrin and its iron complex bearing four phospholipid substituents, in which a trimethylolethane residue connects the two acyl chains (lipid-porphyrins), have been synthesized. The free-base lipid-porphyrin 6a self-organizes in aqueous media to form spherical unilamellar vesicles with a diameter of 100 nm and a uniform thickness of, 10 nm, which corresponds to twice the length of the molecule. In the visible absorption spectrum, the porphyrin Soret band was significantly red-shifted (12 nm) relative to that of the monomer in benzene/MeOH solution due to the excitonic interaction of the porphyrin chromophores. The Pi-A isotherm of 6a gave an area per molecule of 2.2 nm(2), which allowed the estimation of the number of molecules in a single vesicle (2.3 x 10(4)). Double-layered Langmuir-Blodgett (LB) films of 6a on a glass surface exhibited an absorption spectrum identical to that of the 6a vesicles in bulk aqueous solution, and this suggests that they contain similar geometric arrangements of the porphyrin moieties. Exciton calculations on the basis of our structural model reproduced the bathochromic shift of the Soret band well. In the photophysical properties of the 6a vesicles, the characteristics of J-aggregated porphyrins substantially predominate: strong fluorescence and extremely short triplet lifetime. The iron complex 6b with a small molar excess of 1-dodecylimidazole (DIm) also formed spherical unilamellar vesicles (100 nm phi). Scanning force microscopy after evaporation on a graphite surface revealed 6b/DIm vesicles with a vertical height of 19.8 nm, which coincided with the thickness of the double bilayer membranes. The ferrous 6c formed a bis(DIm)coordinated low-spin Fe-II complex under an N-2 atmosphere. Upon addition of O-2 to this solution, a kinetically stable O-2 adduct was formed at 37degreesC with a half-life of 17 h. Distinct gel-phase (liquid-crystal) transitions of the lipid-porphyrin membranes were clearly observed; the free base 6a displayed a higher transition temperature (56degreesC) than the iron complex. Magnetic circular dichroism and infrared spectroscopic studies proved that molecular O-2 coordinates to the self-organized lipid-porphyrinato. iron (II) vesicles in aqueous media.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

The recombinant human serum albumin incorporating a tetraphenylporphyrinatoiron(II) derivative with a covalently linked proximal base (rHSA-FeP) is an entirely synthetic O-2-carrier as a red blood cell substitute. It shows long-term preservation stability of both solution properties and O-2-binding ability over a broad temperature range including room temperature. The physicochemical parameters of the rHSA-Fe(II)P solution ([rHSA]: 5 wt%) stored at 5-40degreesC have remained constant over one year. The in vivo safety of this material was also studied by the survival tests and clinical blood chemistry assays. All rats given the 5 wt% rHSAFe(II)P solution have been alive for 14 days after the infusion. During the observation period, no remarkable reaction was seen in the appearance of the animals. Parameters of the clinical blood serum chemistry were all in the normal ranges, and pathological inspections of the major organs showed no significant infarct in their tissues. Copyright (C) 2003 John Wiley Sons, Ltd.

DOI： 10.1002/pat.240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

incorporation of different structured synthetic hemes, 5,10,15,20-tetraphenylporphyrinatoiron(II) derivetives with a covalently linked proximal base [FeP(1) to FeP(7)], into human serum albumin (HSA), provides seven types of albumin-heme hybrids (HSA-FeP) with different O-2-binding abilities. An HSA host absorbs a maximum of eight FeP molecules in each case. The obtained all HSA-FePs can reversibly bind and release 02 under physiological conditions (in aqueous media, pH 7.3, 37 degreesC) as similar as hemoglobin and myoglobin. The difference in the fence structures did not affect the O-2-binding parameters, however the axial histidine coordination significantly increased the O-2-binding affinity, which is ascribed to the low O-2-dissociation rate constants. The most remarkable effect of the heme structure appeared in the half-lifetime (,tau(1/2)) of the O-2-adduct complex. The dioxygenated rHSA-FeP(4) showed an unusually long lifetime (tau(1/2): 25 hr at 37 degreesC) which is ca. 13-fold longer than that of rHSA-FeP(1).

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

5,10,15,20-Tetrakis[(alpha,alpha,alpha,alpha-o-pivaloylamino)phenyl]porphinatoiron(II) and 5,10,15,20-tetrakis-{[alpha,alpha,alpha,alpha-o-(1-methylcyclohexanoylamino)}phenyl]porphinatoiron(II) complexes bearing a covalently bound 8-(2-methyl-1-imidazolyl)octanoyloxymethyl or 4-(methyl-L-histidinamido)butanoyloxymethyl side-chain [FeRP(B) series: R = piv or cyc, B = Im or His] have been synthesized. The histidine-bound derivatives [FepivP(His), FecycP(His)] formed five N-coordinated high-spin iron(II) complexes in organic solvents under an N-2 atmosphere and showed large O-2-binding affinities in comparison to those of the 2-methylimidazole-bound analogues [FepivP(Im), FecycP(Im)] due to the low O-2-dissociation rate constants. On the contrary, the difference in the fence groups around the O-2-coordination site (pivaloyl or 1-methylhexanoyl) did not significantly influence to the O-2-binding parameters. These four porphinatoiron(II)s were efficiently incorporated into recombinant human serum albumin (rHSA), thus providing the synthetic hemoprotein, the albumin-heme hybrid [rHSA-FeRP(B)]. An rHSA host absorbs a maximum of eight FeRP(B) molecules in each case. The obtained rHSA-FeRP(B) can reversibly bind and release O-2 under physiological conditions (in aqueous media, pH 7.3, 37 degreesC) like hemoglobin and myoglobin. As in organic solutions, the difference in the fence groups did not affect their O-2-binding parameters, but the axial histidine coordination significantly increased the O-2-binding affinity, which is again ascribed to the low O-2-dissociation rates. The most remarkable effect of the heme structure appeared in the half-life (tau(1/2)) of the O-2-adduct complex. The dioxygenated rHSA-FecycP(His) showed an unusually long lifetime (tau(1/2): 25 h at 37 degreesC) which is ca. 13-fold longer than that of rHSA-FepivP(Im).

DOI： 10.1021/bc010067r

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

An amphiphilic (tetraphenylporphyrinato)iron(III) derivative with four alkylphosphocholine groups and a proximal imidazole [(lipidporphyrinato)iron(III); Ia] was self-assembled in phosphate-buffered saline (pH 7.3, [NaCl] = 0.15 M) to form spherical micelles with a diameter of 10 nm. The obtained solution showed a distinct absorption band at 362 mn, which was assigned to the ligand-to-metal [Cl- to iron(III)] charge transfer (LMCT) transition. Light irradiation into this CT band under an Ar atmosphere did not induce any changes in the UV-vis absorption spectrum. On the other hand, the addition of glucose (150 mM) to the solution led to complete photoreduction of the central iron(III) ion, giving a five-N-coordinated high-spin iron(II) complex. It has also been found that a small excess amount of hyaluronic acid ([units] = 100 muM) showed the same effect. The photoreduction was only seen by LMCT irradiation in the presence of the saccharide. It probably occurred via intramolecular electron transfer from Cl- to iron(III), and the produced chlorine radical was scavenged by the saccharide, which prevented a back electron transfer reaction (the quantum yields; ca. 0.007). Interestingly, hyaluronic acid changed the morphology of the Ia assembly from the micelle to a thin fiber. This co-aggregated structure with hyaluronic acid would be responsible for the effective photoreduction of Ia. The viscosity of the fiber solution significantly decreased during the photoreduction, which suggests that radical trapping induces depolymerization of the hyaluronic acid. Laser flash photolysis experiments showed that the reduction and the imidazole association to the iron(II) center are completed within 50 ns after a laser pulse. The photoreduced (lipidporphyrinato)iron(II) fibers can reversibly bind and release O-2 similar to the same fibers which were prepared by chemical reduction using ascorbic acid.

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The electrochemical properties of a synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating 2-[8-{N-(2-methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha -o-pivalamidophenyl)porphinatoiron(II) (FeP) (rHSA-FeP), in aqueous media were investigated using an edge plane pyrolytic graphite electrode modified with didodecyldimethylammonium bromide. The redox behavior of the rHSA-FeP hybrid fits well with thin-film electrochemistry: (1) the redox potential (E-1/2) of the Fe(III)/Fe(II) couple in the hybrid was always constant; however, (2) the peak current and separation increased depending on the scan rate. Because the isoelectric point (pl) of rHSA-FeP is 4.8, its electron density becomes higher with an increase in pH of the aqueous solution (pH 4-9) and the E-1/2 shifts to the anodic side. The counter anions and their concentration affect the E-1/2, suggesting that the counter anions participate in the redox reaction. Compared with that of the naked FeP in aqueous solution, the E-1/2 of rHSA-FeP shifts in the cathodic direction. This indicates that the hydrophobic environment in the albumin host makes the Fe(H) state difficult to oxidize. An increase in the number of the combined FeP molecule(s) in rHSA from 1 to 4 and 8 results in a positive shift of the E-1/2. The first bound FeP probably sits at the outer domain of the albumin structure, and the other seven FeP molecules are in a relatively inner position. (C) 2001 Published by Elsevier Science BN.

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Double-sided porphinatoiron(II)s with polar substituents [R; hydroxy (FeDP(OH)), methoxy (FeDP(OMe)), and acetoxy (FeDP(OAc))] on the 2,2-dimethylpropanoyloxy-fence groups have been synthesized. FeDP(OMe) and FeDP(OAc) formed five-N-coordinated high-spin Fe2+ complexes with an intramolecularly bound axial imidazole in toluene (or CH2Cl2) under an N-2 atmosphere. Upon the addition of O-2, they produced stable O-2 adducts at 25 degreesC; their half-lives in water-saturated toluene (50-77 h) are 2-3 fold longer compared to that of the single-face encumbered porphinatoiron(II) (FeP). Their O-2-binding parameters are almost identical to that of FeDP(H), which has nonpolar substituents on the fences. In contrast, FeDP(OH) showed a significantly low O-2-binding affinity and was immediately oxidized to the Fe3+ state after contact with bubbling O-2 gas. The incorporation of these FeDPs into the human serum albumin (HSA) provided artificial hemoproteins, which can reversibly bind and release O-2 under physiological conditions (in aqueous media, pH 7.3, 37 degreesC like hemoglobin and myoglobin. The half-life of the dioxygenated HSA-FeDP(H) reached 5 h (37 degreesC). This corresponded to a 2.5-fold increase compared to that of HSA-FeP. The time dependences of the absorption changes accompanying the O-2- and CO-rebindings to the HSA-FeDPs after laser flash photolysis were composed of two phases. These observations indicate that the recombination of O-2 and CO to the central Fe2+ ion is affected by the microenvironments around the FeDPs in the HSA structure, e.g. a steric hindrance of the amino acid residue and a difference in polarity. Furthermore, FeDP(H) incorporated into HSA showed a high stability against H2O2.

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The iron complex of tetrakis[2,6-di(alkanoyloxy)phenyl]porphyrin derivative with seven phosphocholine head groups and an N-alkylimidazole side-chain as the proximal base (octopus-porphyrin) has been synthesized. The ferrous complex (7b) was easily dispersed in water to provide a stable colloid. Transmission electron microscopy and scanning force microscopy of the evaporated solution showed spherical micelles with a diameter of 8 nm, which probably consists of a dimer of 7b. These micellar aggregates could reversibly coordinate O-2 at 25 degreesC; and the O-2-binding parameters are also given.

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meso-Tetrakis(alpha,alpha,alpha,alpha -o-pivalamidophenyl iron(II) with an intramolecularly coordinated proximal histidine forms a stable O-2-adduct complex in benzene solution at 25 degreesC; the O-2-binding affinity (P-1/2: 1.7 Torr) is significantly low compared to that of the N-alkylimidazole bound analogue.

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Recombinant human serum albumin (rHSA) incorporating 2- [8-(N-(2-methylimidazolyl))octanoyloxymethyl]-5,10, 15,20-tetrakis(alpha,alpha,alpha,alpha -o-pivalamido)phenylporphinatoiron(II)s (Fe(II)Ps) [rHSA-Fe(II)P] is a synthetic hemoprotein which can bind and release O-2 reversibly under physiological conditions (saline solution [NaCl]: 150 mM, pH 7.3) as do hemoglobin and myoglobin. However, the central ferrous ions of Fe(II)Ps are slowly oxidized to O-2-inactive ferric forms. Based on the W-vis. absorption spectroscopy, the majority of the autooxidized Fe(III)Ps in albumin are determined to be six-coordinate high-spin complexes with a proximal imidazole and a chloride anion, which show ligand-to-metal charge transfer (LMCT) absorption at 330 nm. Interestingly, photoirradiation of this LMCT band under an argon atmosphere led to reduction of the central ferric iron of Fe(III)P, allowing the revival of the O-2-binding ability. The ratio of the photoreduction reached a maximum of 83%, which is probably due to the partial dissociation of the axial imidazole. The same photoirradiation under a CO atmosphere provides the corresponding carbonyl rHSA-Fe(II)P. Laser flash photolysis experiments revealed that the reduction was completed within 100 ns. The quantum yields (Phi) of these photoreductions were approximately 0.01.

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The reaction of nitric oxide (NO) with a synthetic hemoprotein, the recombinant human serum albumin (rHSA) incorporating eight tetraphenylporphinatoiron(II) derivatives bearing a covalently linked axial base (FeP) [rHSA-FeP], has been investigated. The W-vis absorption spectrum of the phosphate buffer solution (pH 7.3) of rHSA-FeP showed maxima at 425 and 546 nm upon the addition of NO. The carbonyl rHSA-FeP, in which FePs are six-coordinate CO-adducts, also moved to the same species after bubbling with NO gas. ESR spectroscopy revealed that the incorporated FePs in the albumin formed six-coordinate nitrosyl complexes; the proximal imidazole moiety does not dissociate from the central iron when NO binds to the trans side. The NO-binding affinity of rHSA-FeP (P-1/2(NO), 1.7 x 10(-6) Torr, pH 7.3, 298 K) was significantly lower than that of FeP itself (P-1/2(NO), 1.8 x 10(-8) Torr in toluene). Kinetically, this arises from the decreased association rate constant (k(on)(NO), 8.9 x 10(8) M-1 s(-1) --&gt; 1.5 x 10(7) M-1 s(-1)). Since NO-association is diffusion controlled, incorporation of the synthetic heme into the albumin matrix appears to restrict the NO access to the central iron(II).

DOI： 10.1021/bc000067b

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Language：Japanese   Publisher：メディカルレビュー社  

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The kinetics of the CO and O-2 binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-(N-(2-methyIimidazolyl))octanoyloxymethyl]-5,10,15,20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s(FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [k(on)(CO)(fast), k(on)(CO)(slow)] were determined to be 4.9 x 10(6) M-1 s(-1) and 6.7 x 10(5) M-1 s(-1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O-2 photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG(double dagger) of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.

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The redox behavior of a synthetic hemoprotein, the recombinant human serum albumin (rHSA), incorporating a tetraphenylporphinatoiron derivative (FeP) [rHSA-FeP] was first evaluated using a graphite electrode modified with didodecyldimethylammonium bromide as a promoter. Compared to that of the naked FeP, the redox potential of the Fe(III)/Fe(II) couple in rHSA-FeP shifts in the positive direction, indicating that the central ferrous ion of FeP becomes more difficult to be oxidized by incorporation into the albumin structure.

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meso-Tetrakis(alpha,alpha,alpha,alpha-o-pivalamidophenyl)porphinato-iron(II) with an intramolecularly bound 2-methylimidazole forms a six-coordinate nitrosyl complex in toluene at 298 K, which is revealed by ESR and IR spectroscopy; the NO-binding affinity (P-1/2) is 1.8 x 10(-8) Torr and the association rate constant (k(on)) is 8.9 x 10(8) M(-1)s(-1).

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A tetraphenylporphyrin derivative having four alkylphosphocholine groups and a covalently-bound axial imidazole (lipidporphyrin) is easily self-organized in water to form spherical micelles or bilayer membranes with phospholipid molecules. The photoinduced electron transfer reactions between lipidporphyrinato-zinc(II)(1) and -iron(III) (2) complexes in these molecular assemblies were studied by fluorescence spectroscopy and laser flash photolysis. A mixture of 1 and 2 (molar ratio: 1/1) produced non-fluorescent micelles. The red-shifted Sorer band absorption, relative to that of the methanolic monomer solution, suggests the formation of the photodeactive complex made of 1 and 2 in the ground state. On the other hand, both chromophores were homogeneously dispersed into the bilayer membrane of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), and the reductive electron transfer from the excited singlet state of 1 to 2 was observed. While this resulted in a significant decrease in the yield of intersystem crossing, the gradual quenching of the excited triplet state of 1 by 2 was also measured. In both cases, the corresponding Stern-Volmer plots showed a linear relationship and yielded quenching rate constants of 1.2 x 10(11) and 6.2 x 10(4) M (-1) s(-1) via the excited singlet state and the triplet state, respectively. In the presence of excess triethanolamine as a sacrificial reagent, the intermolecular electron transfer became partly irreversible, giving an intramolecularly imidazole-coordinated Fe(II) complex which is capable of reversibly binding dioxygen like hemoglobin. (C) 2000 Elsevier Science S.A. All rights reserved.

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Human serum albumin (HSA) incorporating synthetic hemes, the tetrakis(o-pivalamido)phenyl-porphinatoiron(II) derivative (FeP), is an artificial hemoprotein (HSA-FeP) which is able to reversibly bind and release dioxygen under physiological conditions (in aqueous media, pH 7.4, 37 degrees C) like hemoglobin and myoglobin. Physiological responses to exchange transfusion with HSA-FeP solution [[HSA], 5 g/dL; FeP/HSA, 4 (mol/mol)] into rats after hemodilution and hemorrhage (Hct, about 10%) has been evaluated. The declined mean arterial pressure (MAP) and blood flow after a 70% exchange with HSA and the further 40% bleeding of blood were significantly recovered up to about 90% of the baseline values by the injection of HSA-FeP. Furthermore, the renal cortical O-2-tensions and skeletal. tissue O-2-tensions were also increased, indicating the in vivo O-2-delivery of HSA-FeP. Autoxidation of ferrous Fe(II)P to ferric Fe(III)P was retarded in the blood stream; the half-lifetime of the dioxygenated FeP [tau(1/2)(O-2)] in vivo was 4.1 h [cf. 1.0 h (in vitro)]. It has been found that autooxidized Fe(III)P was certainly reduced in the whole blood suspension. Physiological concentrations of ascorbic acid continuously provided by red blood cells probably rereduces Fe(III)P, leading to the apparent long lifetime of the dioxygenated species of FeP.

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Photoirradiation into the LMCT band (lambda(max): 362 nm) of self-assembled amphiphilic tetraphenylporphyrinato-iron(III) (lipidporphyrinato-iron(III)) chloride with hyaluronic acid leads to reduction of the central ferric ion in saline solution (pH 7.4, 25 degrees C); the obtained lipidporphyrinato-iron(II) can reversibly bind and release dioxygen.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

Lipidporphyrinatoiron(II) complexes are tetrakis(o-substituted)phenylporphinatoiron(II) derivatives which can be easily dispersed in water by molecular assembling. The most remarkable aspect of lipidporphyrinatoiron(II) assemblies is their reversible binding of dioxygen under physiological conditions tin aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. in these structures the O-2-binding properties are largely influenced by the molecular environment around the coordination site. Tetrakis(o-pivalamido)phenylporphinatoiron(II) with a covalently linked axial imidazole (lipidporphyrinatoiron(II), 1) is incorporated into recombinant human serum albumin (rHSA), providing a totally synthetic O-2-carrying hemoprotein (rHSA-1). Electrospray ionization mass spectrometry revealed the molecular mass of this non-covalent albumin-porphyrin hybrid. The O-2 rebinding after laser flash photolysis represented a three-phase decay, suggesting that each porphyrin is embedded into different cavities in the albumin structure. On the other hand, amphiphilic Lipidporphyrinatoiron(II) with four alkylphosphocholine chains (2) is self-organized in aqueous solution to produce bimolecular fibers with a uniform thickness of 10 nm. This fiber also gave a stable O-2 adduct, and the O-2 rebinding after laser flash irradiation showed monophasic kinetics. Up to 20 vol% of methanol, which is a critical concentration for fiber formation, the morphology was gradually dissociated into spherical micelles, and the stability of the dioxygenated species suddenly decreased to 10% of that of the fibers. Copyright (C) 2000 John Wiley & Sons, Ltd.

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Recombinant human serum albumin (rHSA) was dimerized by bis(maleimido)hexane through the free thiol at Cys34. The molecular mass of the dimer [(rHSA)(2)] was determined by native PAGE electrophoresis and MALDI-TOFMS spectrometry. As expected, the colloid osmotic pressure was only half that of the monomeric rHSA solution. Incorporation of (2-[8-{N-(2-methylimidazolyl)}-octanoyloxymethyl]-5,10,15,20-tetrakis(o-pivalamido)phenylporphinato)iron(II)s (FePs) into the hydrophobic cavities of the rHSA dimer provides a synthetic hemoprotein, [(rHSA-FeP)(2)], which can reversibly bind and release dioxygen under physiological conditions tin aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. A maximum of 16 hemes (FePs) were incorporated into the (rHSA)(2) structure. On the basis of the isoelectric focusing measurement, the surface charge distributions of the (rHSA)(2) and (rHSA-FeP)(2) are identical to that of rHSA. The O-2-binding affinity (P-1/2: 30 Torr at 37 degrees C) and O-2-association and -dissociation rate constants of (rHSA-FeP)(2) (k(on), 2.4 x 10(7) M-1 s(-1); k(off), 4.7 x 10(2) s(-1)) satisfy the requirements for a synthetic O-2 carrier as a red cell substitute.

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A 5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivalamidophenyl)porphinatoiron(II) derivative bearing a covalently linked axial imidazole via a rigid benzoyloxy spacer (8c) has been synthesized. The O-2(-) and CO-binding affinities of 8c (P-1/2(O2): 213 Torr, P-1/2(CO): 0.17 Torr in toluene at 25 degrees C) were significantly lower than those of a similar analog having a flexible omega-imidazolylalkyl group (9c). Kinetically, the low binding affinities of these gaseous molecules are attributed to the high dissociation rate constants. The stretching frequency of the CO bound to the central Fe(II), however, showed exactly the same value (v(CO): 1964 cm(-1)) for both complexes. These results suggested that the intramolecular imidazole coordination was not disordered by the rigid benzoyloxy spacer in 8c. We concluded that the low O-2(-) and CO-binding affinities of 8c were caused by the low alpha-basicity (pK(a)) of the imidazole ring. (C) 1999 Elsevier Science S.A. All rights reserved.

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2-[8-{N-(2-Methylimidazolyl)}octanoyloxymethyl]-5,10,15,20-tetrakis(o-pivalamido)phenylphorin-atoiron(II)s (FePs) were incorporated into hydrophobic cavities of recombinant human serum albumin (rHSA), providing a totally synthetic O-2-carrying hemoprotein (rHSA-FeP). An rHSA host absorbs maximally eight FeP molecules. Solution properties of the obtained albumin hybrid [[rHSA] = 5 wt%; FeP/HSA = 1-8 (mol/mol)] are almost identical to those of the rHSA itself; the specific gravity is 1.013 and the viscosity is 1.1 cP. Circular dichroism spectroscopy and isoelectric focusing measurement revealed that the second-order structure and surface charge distribution of rHSA were always constant independent of the binding numbers of FeP. Hydrophobic interaction is probably a major molecular force of the incorporation of this synthetic heme. rHSA-FeP can bind and release dioxygen reversibly under physiological conditions (in aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. Its O-2-coordination structure was evaluated by resonance Raman spectroscopy. The O-2 rebinding after the laser flash photolysis showed three-phases decay, which were analyzed by triple-exponential kinetics. The O-2-binding affinity and O-2-association and -dissociation rate constants of rHSA-FeP satisfy the initial clinical requirements for O-2 infusion as a red cell substitute.

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Amphiphilic tetraphenylporphyrinato-metal (Zn(II), Fe(II)) derivatives with four alkylphosphocholine chains and an intramolecular coordinated axial imidazole (lipidporphyrins) produced stable colloidal solutions in water. Electron microscopy showed rodlike fibers with a uniform thickness of 10 nm, which corresponds to a double length of lipidporphyrin. The obtained fibers had some micellar characteristics and were sensitive to neither addition of electrolyte (for example, NaCl) nor change in pH. Scanning force microscopy (SFM) revealed evaporated fibers with a height of 2.8 nm on graphite. In the center of the fiber, there is probably a tetragonal tube constructed by densely packed porphyrin planes. The red shift in the broadened Soret band absorption by exciton interaction and the photophysical properties of the Zn(II) complex fibers suggested the formation of porphyrin J-aggregates. Furthermore, the first SFM images of the porphyrin fibers under liquid conditions are given. A Benesi-Hildebrand analysis revealed that the unit aggregate number for the Zn(II) complex was four, supporting a tetramer repeating structure. Fe(II) complex fibers, on the other hand, produced a kinetically stable O-2 adduct reversibly at 25 degrees C. The coordination structures of the axial imidazole and O-2 molecule in the Fe(II) complex have been clarified by resonance Raman spectroscopy. The O-2-binding equilibrium and kinetic parameters were also evaluated.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：American Chemical Society  

The structure, physicochemical properties, O2-binding ability of lipidheme vesicles and hemoglobin (Hb) vesicles were evaluated. The solution properties of lipidheme vesicles and Hb-vesicles are low in comparison to blood, due to their small sizes. The O2-binding rate of Hb is affected by encapsulation. In fact, the O2-association of Hb vesicles is slower than that of Hb itself, but faster than that of the red blood cell. Since the lipidheme locates in the bilayer membrane, lipidheme shows a faster O2-binding rate constant than Hb. Inspite of structural differences, both materials can act as dioxygen infusion.

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A tetraphenylprophyrinatoiron(II) derivative (FeP) with a covalently linked axial base, 2-[8-{N-(2- methylimidazolyl)}octanoylxymethyl}-5,10,15,20- tetrakis(α,α,α,α-o- pivalamido)phenylprophyrinatoiron(II), was efficiently incorporated into the human serum albumin (HSA) molecule providing an artificial hemoprotein which is able to bind and release dioxygen reversibly under similar physiological conditions as hemoglobin. The imidazole intramolecularly coordinated to the central iron plays a crucial role in the formation of a stable Fe-O2 complex. The physicochemical and the physiological responses of the HSA-FeP to exchange transfusion with the HSA-FeP into hemorrhagic shocked model of anesthesized rats are reported.

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The photophysical and photochemical properties of 5,10, 15,20-tetrakis{alpha,alpha,alpha,alpha-o-[2',2'-dimethyl-20'-((2"-(trimethylammonio)ethyl) phosphonatoxy)alkanamido]phenyl}porphinatozinc(II) (zinc lipidporphyrins, ZnLPs (C-10, C-18)) have been studied in homogeneous DMSO solution and compared with those of 5, 10,15,20-tetrakis{alpha,alpha,alpha,alpha-o-pivalamidophenyl}porphinatozinc(II) (ZnTpivPP) and tetrakis-phenylporphinatozinc(II) (ZnTPP). The fluorescence quantum yields of the ZnLPs were lower than that of ZnTPP, but their fluorescence lifetimes were relatively long. The electron transfer reactions from the photoexcited states of these Zn porphyrin complexes to several quinone derivatives were evaluated by fluorescence spectroscopy and laser hash photolysis. The efficiencies of oxidative quenching of the excited porphyrins via a dynamic process were significantly decreased by the presence of the bulky substituents on one side of the porphyrin macrocycle. This steric effect of the porphyrin side-chains was quantitatively examined by the Marcus classical treatment. (C) 1999 John Wiley & Sons, Ltd.

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Incorporation of tetrakis(o-pivalamido)phenylporphyrinatoiron(II) derivatives with a covalently linked axial imidazole (FeP) into human serum albumin (HSA) provides a new type of artificial hemoprotein (HSA-FeP) that binds and releases dioxygen reversibly under physiological conditions (in aqueous media, pH 7.4, 37 degrees C) and in a fashion similar to hemoglobin and myoglobin. The HSA host adsorbs a maximal eight FeP molecules, and their stepwise equilibrium constants (K-1-K-8) range from 1.2 x 10(6) to 1.3 x 10(4) M-1. The major binding sites of the synthetic hemes are identical to those of hemin, bilirubin, and long-chain fatty acids. The red-colored solution of HSA-FeP was stored for three months at 4 degrees C and could be kept as a freeze-dried powder for more than six months. The solution properties [[HSA]: 5 wt %, FeP/HSA = 1-8 (mol/mol)] satisfy the physiological requirements for dioxygen infusion for potential clinical use; the specific gravity is 1.013, and the viscosity is 1.1 cP. Mixing the solution with human blood does not induce any coagulation and precipitation. On the basis of the gel permeation chromatography, CD spectroscopy, and IEF measurements, the molecular size, second-order structure, and surface charge distribution of the HSA-FeP conjugate are constant and independent of the binding numbers of heme molecules. Furthermore, the O-2-coordination structure of FeP embedded into certain hydrophobic domains of the albumin was confirmed by resonance Raman spectroscopy.

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Bolaamphiphilic tetraresorcinolporphyrins with eight long side chains (octopusporphyrins) and their metal complexes form monolayered assemblies in bulk aqueous solution. The nano-structure, the photoinduced electron transfers and the O-2 coordination of these octopusporphyrin assemblies are described. In the micellar fibers of 1a and 1b, a unique spherical arrangement of eight methyl groups on both sides of the porphyrin ring plane provides hydrophobic porphyrin centers which align in a string of pearls. Exciton calculations indicated a tilt stacking porphyrins arrangement with a separation of 11 Angstrom. These fibers fluoresced strongly electron transfer reaction was therefore observed between the porphyrin center and hydrophobic quenchers as well as hydrophilic quenchers. The fibers were also active as photocatalysts in the reduction of dimethylviologen by triethanolamine. Octopusporphyrins with different metal centers can also produce fibrous aggregates, for example, H2P/Zn(II)P and ZnOP/Fe(III)P couples. The fluorescence quenching of Zn(II)P in the ZnOP/Fe(III)P hybrid fibers can be ascribed to the intermolecular electron transfer within the fibers. In H2P/Zn(II)P couple, excitation energy transfer from excited Zn-1*p to H2P occurred after photoexcitation. Octopusporphyrin with four dialkylglycerophosphocholine groups on both sides of the ring plane (2b) forms spherical unilamellar vesicles. Based on cryomicroscopy, a white line was observed with a diameter of 15 Angstrom in the middle of the membrane which are obviously a porphyrin layer with low molecular packing. The octopusheme (2c) vesicles prepared in a similar manner with 20-fold excess molar coexistence of 1-dodecyl-2-methylimidazole (DMIm) can bind and release oxygen reversibly at 25 degrees C. Moreover, water-soluble octopusporphyrin (3a) produced fluorescent and non-fluorescent monolayer assemblies anion exchange of the head groups, e.g. 3a with um perchlorate showed planar sheets. An exciton calculation is consistent with a two-dimensional arrangement with porphyrin separations Of 25.6 and 17.4 Angstrom in the x- and y-directions, respectively. External addition of negatively charged electron accepters, naphtoquinone sulfonate and anthraquinone sulfonate, led to partial quenching of the fluorescence of the central porphyrin layer. The results have been evaluated using equations derived for this special quenching. (C) 1998 John Wiley & Sons, Ltd.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Water-soluble tetraresorcinolporphyrins with eight omega-pyridinium alkyl chains (octopusporphyrins) formed fluorescent and nonfluorescent monolayer assemblies by anion exchange of the headgroups. Electron microscopy of the evaporated solution of the octopusporphyrin having 11-pyridinium-undecanoyl side chains (1) with sodium perchlorate showed planar sheets. The uniform thickness of the layer was 4.0 +/- 0.5 nm, corresponding to monomolecular platelets. An exciton calculation on the basis of the red shift of the Soret band (6 nm) is consistent with a two-dimensional arrangement with porphyrin separations of 25.6 and 17.4 Angstrom in the x and y directions, respectively. Organic dianions such as anthraquinone-2,6-disulfonate (AQDS(2-)) were more effective than perchlorates or iodides for aggregate formation. Arrays of a 1:3 complex of 1/AQDS(2-) produced a curvature to yield nonfluorescent vesicles. The introduction of dimethyl groups at the bottom of the alkyl chains (octopusporphyrin 2) did not lead to enhanced aggregation, while the octopusporphyrin with long 2,2-dimethyl-C-20-pyridinium chains (3) formed fluorescent fibers without assistance of special anions. The electron-transfer reactions of the 1 platelets with perchlorates, in which the relative fluorescence intensity was 30% of the monomer, were investigated. External addition of negatively charged electron accepters, 1,2-naphthoquinone-4-sulfonate (NQS(-)) and anthraquinone-2-sulfonate (AQS(-)), led to partial quenching of the fluorescence of the central porphyrin layer. In both cases, the corresponding Stern-Volmer plots showed plateaus at sufficiently high concentration of the quinones. The results have been evaluated using equations derived for this special case of dynamic quenching by an electrostatically bound quencher. Binding constants of 3.4 x 10(4) and 1.7 x 10(5) M-1 and electron-transfer constants of 5 x 10(9) and 1.3 x 10(9) s(-1) have been calculated for NQS(-) and AQS(-), respectively.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Resonance Raman spectra with 457.9 nm laser excitation have been recorded for the deoxy and oxy complexes of ortho-substituted tetraphenyl- and tetranaphthyl-porphyrinatoiron(II) derivatives with a covalently linked proximal imidazole. The intramolecular imidazole co-ordination gives high-spin iron(II) states in the deoxy forms of these complexes, which are revealed by the appearances of the indicative nu(s) bands of the porphyrin ring at 367-375 cm(-1). Their Fe-N (imidazole) stretching modes were directly detected at 201-223 cm(-1), and are discussed in relation to the Fe-N (imidazole) bond strength and the geometry of the imidazole co-ordination. As a result, a significant tilting of the imidazole ring plane from the Fe-N (imidazole) vector is proposed for the double-side encumbered derivatives. The dioxygenation was monitored by the upshift of the porphyrin skeletal band (nu(4)). The dioxygen adducts of the double-side encumbered porphyrins showed high iron-dioxygen stretching frequencies (579 and 580 cm(-1)) relative to those of oxyhemoproteins and other dioxygenated porphyrinatoiron(II) complexes generally observed in the range 568-573 cm(-1). These high frequencies are considered to reflect the decreased FeO-O angle induced by the narrow eater-linked cavity. For complexes of 5,10,15,20-tetrakis(o-pivalamidophenyl)porphyrinatoiron(II) with a covalently attached imidazolyl group at the beta-pyrrole position the nu(Fe-N-epsilon) and nu(Fe-O-2) frequencies were found to be fairly similar to those of the same complexes with externally added imidazole ligands. This indicates that the covalent linkage between the imidazole and the porphyrin periphery is flexible and long enough to allow the formation of stable O-2-adduct species.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Intramolecular N-imidazole coordinated [5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivaloyloxynaphthyl)porphinato]iron(II) forms a stable dioxygen adduct in toluene at 25 degrees C; the sterically regulated coordination of the axial base leads to the low electron donation from the central iron to the bound O-2.

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Language：English   Publisher：ポルフィリン研究会  

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Language：English   Publisher：ポルフィリン研究会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Micellar fibers and monolayers made of amphiphilic, cationic porphyrins are stabilized in bulk water by poly( allylamine) at pH values close to the pk(a) of the amines. Anionic polyelectrolytes, e.g., poly(vinyl sulfate), can also be used, but in order to prevent precipitation one must use a large polymer excess and start with small assemblies. The polyelectrolyte additions not only prevent precipitation of the large molecular assemblies with molecular weights far above 10(6) Da, but also draw back any monomers into the assemblies. Small absorption changes in 3 ns flash photolysis experiments became detectable only in the polymer-stabilized solutions. (C) 1998 Academic Press.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Human serum albumin (HSA) incorporating synthetic tetraphenylporphinatoiron(II) derivatives (FeP1 or FeP2) can bind and release oxygen reversibly under physiological conditions (in aqueous media, pH 7.4, 37 degrees C). The maximal binding ratio of FeP1/HSA was estimated to be eight, and the stepwise equilibrium constants for FeP1 binding to HSA (K-1-K-8) ranged from 1.2x10(6) to 1.3x10(4) M-1. The major binding sites of FeP1 are presumably identical to those of hemin, bilirubin and long-chain fatty acids. The O-2-binding ability of the HSA-FeP can be regulated by changing the molecular structure of the incorporated hemes. The half-lifetime of the O-2-coordinated FeP2 in HSA was significantly longer than that of HSA-FeP1.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Tetrakis(aminophenyl)porphyrin with four bixin side chains in statistical orientation with respect to the porphyrin plane and its zinc(II) complex produced stable colloidal solutions in water at pH 9, Cryoelectron microscopy showed spherical unilamellar vesicles with diameters ranging from 30 to 120 nm and a membrane thickness of 4.7 +/- 0.5 nm, corresponding to a monomolecular layer with the bixin side chains tilted by 30 degrees. The stiff vesicle membranes were not sensitive to the addition of sodium chloride and survived adsorption to dry solid surfaces. Furthermore, the vesicles were totally polymerized by visible light, These polymer vesicles remained intact in 95% ethanol, and scanning force microscopy (SFM) images showed perfectly spherical shapes on graphite. A slow collapse is only observed on hydrated mica, The tetrabixinporphyrin also produced stable monolayers on water surface which polymerized upon irradiation. Combination of the polyene vesicle with an electron-accepting porphyrin, namely a synthetic guanidinium porphyrin or 2-anthraquinone sulfonate, led to light-induced long-lived charge separations.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A hydrophobic tetraphenylporphyrinatoiron(II) derivative bearing a covalently bound axial imidazole [Fe(II)P] was efficiently and noncovalently bound into human serum albumin (HSA) up to an average of eight Fe(II)P molecules per HSA molecule. The aqueous solutions of the HSA-Fe(II)P complex provided a reversible and relatively stable oxygen adduct under physiological conditions (pH 7.4 and 37 degrees C). The half-life of the oxygen adduct (tau(1/2)) was 1 h at 37 degrees C in an air atmosphere. With Fe(II)TpivPP (the so-called ''picket-fence heme'') having no axial base, an oxygenated HSA-Fe(II)TpivPP complex was obtained using a 20-fold molar excess of 1,2-dimethylimidazole, but the tau(1/2) was very short (ca. 10 min at 37 degrees C). The oxygen affinity [P-1/2(O-2)] and oxygen transporting efficiency (OTE) of HSA-Fe(II)P at 37 degrees C were 30 Torr and 22%, respectively. Furthermore, the oxygen-binding and dissociation rate constants (k(on), and K-off) are extremely high in comparison with those of hemoglobin. The HSA molecule binding eight Fe(II)P molecules can transport about 3.4 mL/dL of oxygen under physiological conditions, corresponding to about 60 % of the oxygen transporting amount of human blood.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：VCH PUBLISHERS INC  

In aqueous media substituted protoporphyrins and amphiphilic tetraphenylporphyrins aggregate to fibers, tubules, helical ribbons and sheets. Six different types are presented, characterized by the excitonic shifts in the electronic absorption spectra. On excitation, the aggregates from octopusporphyrin show fluorescence and population of the triplet state. In other aggregates fluorescence, triplet state or both are effectively quenched.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Symmetrical tetraresorcinolporphyrin with eight alkylphosphocholine side chains (octopus porphyrin) and its zinc(II) complex were dispersed in aqueous media to give a stable colloidal solution. Electron microscopy showed either planar monolayers or micellar fibers with a uniform thickness of 7 nm. A unique spherical arrangement of eight methyl groups on each side of the porphyrin center provides fat droplet like centers which align in a string of pearls in an extremely open fiber structure. Exciton calculations on the basis of a long wavelength shift of the Soret bands (7-8 nm), line broadening, and electron micrographs indicated a tilt stacking arrangement with a porphyrin separation of 11 Angstrom. The octopus porphyrin fibers fluoresced strongly, and laser flash photolysis lead to electron transfer from the porphyrin to hydrophobic phenyl-p-benzoquinone as well as to hydrophilic 1,2-naphtoquinone-4-sulfonate and dimethylviologen acceptors. The fibers were also active as photocatalysts in the reduction of dimethylviologen to the corresponding radical by amines.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本人工臓器学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUTHIG & WEPF VERLAG  

Amphiphilic metalloporphyrins assemble in water to form supramolecular fibers, which have been characterized by transmission electron microscopy. Loose octopus porphyrin fibers can be doped with hydrophobic electron acceptors, metalloporphyrin monomolecular sheets are crystalline. Charge separation occurs in amino porphyrin fibers without added electron acceptors. Bolaamphiphilic porphyrins with four pyridinyl or methyl pyridinium groups in beta-pyrrolic positions have been synthesized. The regioisomer mixture is very soluble in water (congruent to 0.1 M) and is ideally suited to form heterodimers with negatively charged ms-tetrasubstituted porphyrins. Bimetallic porphyrinate pairs are described. The zinc complex is stable down to pH 1.0. Regioisomer II forms well-defined molecular monolayer leaflets in bulk water at pH 2.5. The surface structure of such monolayers is discussed. It consists of a large cationic plane and hydrophobic edges. Possible applications are discussed shortly.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

A protoporphyrin IX derivative having three long alkyl chains and an axial imidazole (lipidprotoporphyrin) was homogeneously embedded into the bilayer membrane of the phospholipid vesicle. The O-2-binding affinity of the lipid-protoheme was affected by the phase transition of the bilayer membrane, reflecting the change in the O-2-association rate constant.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

An amphiphilic tetraphenylporphyrinatometal (Zn, Fe) derivative having four dialkylglycerophosphocholine groups on one side of the ring plane (lipidporphyrin) was easily dispersed in an aqueous medium to give a red, stable dispersion. Based on electron microscopy, lipidporphyrins themselves produced spherical unilamellar vesicles with a diameter of ca. 100 nm. The thickness of the membrane (9.5 +/- 0.5 nm) corresponded to a bilayer of the lipidporphyrin (4.6 nm); the ratio of the lipidporphyrin molecules in the outer and inner layers was 1.57. The lipidporphyrinatozinc(II) vesicle displayed some characteristics of a J-aggregate: (i) a red-shifted Soret band (425 --&gt; 438 nm), (ii) no fluorescence quenching, and (iii) a reduced triplet-state lifetime. Simple exciton calculations indicate that the porphyrin squares located in the outer and inner layers formed a J-aggregate with an average angle of 47 degrees. A vesicle composed of the lipidporphyrinatoiron(II) coordinated with an alkylimidazole reversibly formed a stable dioxygen adduct. The O-2-binding equilibrium and kinetic parameters were determined. This vesicle has the ability to act as a totally synthetic O-2 carrier under physiological conditions (pH 7.4, 37 degrees C).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The amphiphilic tetraphenylporphin derivative having four dialkylglycerophosphocholine groups on both sides of the ring plane (octopus-porphyrin) forms spherical monolayered vesicle membranes in water with diameters of ca, 100 nm; the vesicle constituted by octopus-porphyrinatoiron(II)/1-dodecyl-2-methylimidazole (DMIm) can reversibly bind dioxygen at 25 degrees C.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：化学同人  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

5,10,15,20-Tetrakis(o-pivalamidophenyl)porphyrins (TPVP) and their iron(II) derivatives bearing a proximal imidazole covalently bound at the 2-(beta-pyrrolic) position have been synthesized. The visible absorption maxima (lambda(max)) of the 2-substituted TPVP with an electron withdrawing group attached is shifted toward the red region (7-11 nm) compared with that of the original TPVP. The iron(II) complexes having an imidazolyl group at the beta-pyrrolic position were five coordinated species with an intramolecularly bound base under argon and reversibly formed a stable dioxygen adduct in response to O-2-pressure in toluene at 25 degrees C. These molecules act as efficient dioxygen carrying molecules. The kinetics of O-2 binding to the 2-substituted Fe(TPVP) complexes are described.

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本血液代替物学会  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Marcel Dekker Inc  

Proof that fat microspheres may act as vehicles of synthetic hemes is presented. The characteristics of the lipid heme microsphere (LHM) are given. The oxygen carrying ability of the LHM is studied in dogs undergoing hemorrhagic shock.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

New protoporphyrin IX derivatives having four amphiphilic and/or lipophilic alkyl chains (lipidporphyrins) were synthesized. The lipidporphyrins were efficiently incorporated into the bilayer membrane of phospholipid vesicle without a stacked arrangement. Homogeneous dispersing property of the lipidporphyrins in the bilayer membrane was maintained after polymerization of the phospholipid molecules.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本人工臓器学会  

In vivo O₂-transporting ability of totally synthetic artificial red cell (lipid-heme-microsphere) is described. O₂-Binding affinity of the lipid-heme-microsphere (P_1/2(O₂): ca. 40 Torr at 37°C) was slightly lower than that of red cell (human blood) and its O₂-association and-dissociation rate constants were larger than those of hemoglobin. O₂-Solubility of the lipid-heme-microsphere dispersion was estimated. Highly O₂-dissolved aqueous solution can be prepared by the prescription of the lipid-heme-microsphere. The totally synthetic artificial red cell suspension was intravenously injected into hemorrhagic beagles (8kg). Circulation life-time of the each constituent of this system and O₂-consumption by the lipid-heme was determined. These results suggest that the lipid-heme-microsphere has an ability to act as an efficient O₂-carrier in blood stream.

DOI： 10.11392/jsao1972.23.849

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Triglyceride microsphere emulsified with phospholipid derivative of heme (5,10,15,20-tetrakis[alpha,alpha,alpha,alpha-o-[2,2-dimethyl-20-[2-(trimethylammonioethoxy) phosphonatoxy]eicosanamido]phenyl]porphinatoiron(II); lipidheme) provides a totally synthetic artificial red cell (lipidheme-microsphere; LH-M). Its structure, solution properties and O-2, binding ability are described. The particle diameter of the LH-M was ca. 90 nm phi elucidated by electron microscopy. Viscosity of the LH-M suspension (similar to 1.5 cP) was much lower than that of human blood and the viscosity of mixed system of LH-M/human blood (1/1(v/v)) was 2.5 cP. Specific gravity, osmotic pressure, and colloid osmotic pressure of the LH-M suspension also satisfied the physiological needs. The LH-M can bind O-2 reversibly in response to O-2 pressure (P-50(O-2): 41 torr (pH 7.4, 37 degrees C)). O-2 solubility of the LH-M was more than that of human blood caused by its high heme concentration.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

We have succeeded in synthesizing a new type of totally artificial oxygen carrier which was produced by covering oil droplets (microsphere) with synthetic hemes (LH-M). We studied its oxygen-transporting ability in hemorrhagic dogs.
Four beagles weighing about 8 kg were studied. Under controlled ventilation, exchange-transfusion of 30 ml/kg was carried out. Cardiac output, hemoglobin and LH-M concentration in the blood, and blood gas were measured to 5 hours after intravenous injection of LK-M solution.
LH-M delivered 15.7 to 22.3 ml/min (11 to 16 %) of oxygen to the tissue and 5.5 to 8.2 ml/min (11 to 17 %) of oxygen was consumed from LH-M to 5 hours after intravenous injection. Its half-life time in the blood stream was about 12 hours. It was confirmed that LK-M transported oxygen and released it to the tissue in vivo.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Two types of totally artificial oxygen carriers were produced (1)by embedding synthetic lipidhemes (as oxygen carriers) in bilayers of liposomes as vehicles of lipidhemes(L/H) and (2)by covering clinically available fat droplets (triglyceride microspheres) with synthetic lipidhemes(LHM). Fat droplets were used as vehicles of lipidhemes. Their oxygen carrying ability in vivo was examined in beagles undergoing hemorrhagic shock. L/H delivered 15.7 to 19.2 % of total oxygen delivery. From 12.7 to 24.4 % of total oxygen consumption was from L/H. LHM delivered 11.6 to 7.3 % of total oxygen delivery. From 13.1 to 16.4 % of total oxygen consumption was from LHM.
These totally synthetic red blood cell substitutes can be candiates for futere clinical testing.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Protoporphyrin IX derivatives having two alkylphosphocholine groups (lipid-porphyrins) forms stable fibrous aggregates in aqueous medium; fibres have been spontaneously incorporated into the bilayer of the phospholipid vesicle.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

A protoporphyrin IX derivative having a phospholipid residue (lipid-porphyrin) was synthesized. The lipid-porphyrin itself formed an oval multilamellar vesicle in an aqueous medium. The spectral features of the lipid-porphyrin vesicle indicated a stacked configuration of the porphyrin moiety.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Double-sided porphyrinatoiron(II) complexes bearing covalently bound axial imidazole, 5-[2-(5-imidazolylvaleryloxy)-6-(pivaloyloxy)phenyl]-10,15,20-tris[2,6-bis(pivaloyloxy)phenyl]porphyrinato-iron(II) and 5-[2-(3,3-dimethylbutyryloxy)-6-(5-imidazolylvaleryloxy)phenyl]-10,15,20-tris[2,6-bis(3,3-dimethylbutyryloxy)phenyl]porphyrinatoiron(II), have been synthesized. On the basis of their absorption and H-1 NMR spectra, the axial imidazole group is co-ordinated. The complexes reversibly form stable dioxygen adducts in toluene at 25-degrees-C, and the kinetics of binding of 02 and CO has been investigated. When embedded in phospholipid unilamellar vesicles, the complexes possess the ability to transport dioxygen in an aqueous medium. The binding affinity of the pivaloyloxy derivative [P1/2(O2) = 27 Torr] is equal to that of a red blood cell suspension and the half-life of the dioxygen adduct formed was 1.5 d under physiological conditions (pH 7.4, 37-degrees-C).

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

New double-sided porphyrinatoiron(II) complexes having a polar cavity which includes an amide moiety (5,10,15-tris[2,6-bis(3,3-dimethylbutyryloxy)phenyl]-20-[2-[N-acetylvalyloxy]-6-(3,3-dimethylbutyryloxy)phenyl]-porphinatoiron(II) (1b) and 5,10,15-tris[2,6-bis(3,3-dimethylbutyryloxy)phenyl]-20-[2-[3,5-bis(acetamido)ben-zoyloxy]-6-(3,3-dimethylbutyryloxy)phenyl]porphinatoiron(II) (2b)) were synthesized. H-1 NMR spectroscopy indicated that the amide residues are located on the porphyrin ring plane. The 02 and CO binding affinities of 1b and 2b were higher than those of 5,10,15,20-tetrakis[2,6-bis(3,3-dimethylbutyryloxy)phenyl]porphinatoiron-(II) (4b), in response to the local polarity in the cavity. The polar amide residue resulted in a decreased O2 dissociation rate. Thermodynamic parameters for the gaseous ligand bindings to the 2b complex were also determined.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Amphiphilic tetraphenylporphyrins having four alkyl phosphocholine groups on each side of the ring plane (octopus-porphyrins) form fibrous aggregates in dilute aqueous solution; the vesicles, constituted by iron(II) derivative-1-dodecyl-2-methylimidazole (DMIm), have the ability to bind dioxygen reversibly in aqueous medium.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Amphilphilic tetraphenylporphin derivatives having four dialkylglycerophosphocholine groups on the ring plane (lipid-porphyrins) form spherical unilamellar vesicles of diameter ca. 100 nm in water; the vesicles composed of lipid-porphyrinatoiron(II)-1-dodecylimidazole (DIm), can bind dioxygen reversibly in aqueous medium.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：高分子学会  

DOI： 10.1295/kobunshi.42.422

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Other Link： https://jlc.jst.go.jp/DN/JALC/00020802219?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本人工臓器学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Synthesis and characterization of two amphiphilic tetraphenylporphinatoiron complexes having a glycerophosphocholine or an alkyl phosphoserine group are described. These porphinatoiron(II) complexes with 1-dodecyl-2-methyl-imidazole (L2MIm) were efficiently embedded in the bilayer of a phospholipid vesicle due to their high compatibility with lipids, similar to the heme substituted with four alkyl amphiphilic chains (lipid-heme). Oxygen transporting ability of the phospholipid vesicles embedded with hemes were similar to that of hemoglobin (Hb) in red blood cells.

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本人工臓器学会  

Solution properties and O₂ transporting ability of lipidheme-microsphere composed of oil droplet covered with lipidheme (totally synthetic artificial red cell) are described. Electron micrographs indicated microsphere formation and its particle diameter was controlled from 30 to 200nm. The suspension was characterized by its low viscosity (1, 2cP), specific gravity (1.001), and controlled oncotic pressure. The lipidheme-microsphere bound oxygen reversibly and the P_1/2(O₂) was 41 torr at 37°C. O₂ solubility of the lipidheme-microsphere was more than that of human blood caused by its high heme concentration. Exchange transfusion with the suspension to dogs weighing ca. 8kg was carried out and circulation half life in blood stream was determined (12hr). This result suggests that the lipidheme-microsphere has potential to act as an efficient oxygen carrier in blood stream.

DOI： 10.11392/jsao1972.22.550

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lipophilic heme (1-laurylimidazole-ligated 5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-omicron-pivalamidophenyl)porphinatoiron(II) complex) is solubilized in lipid (triglyceride) at high concentrations and emulsified with a phospholipid in physiological salt solution, giving a deeply red-colored suspension of lipid microspheres (approx. 250 nm in diameter). The heme forms an oxygen adduct in a similar manner as oxyhemoglobin and the lipid microspheres take up and release oxygen reversibly at 37-degrees-C in the aqueous medium. The oxygen-transporting ability is comparable with that of the red blood cell. Intravenous injection of thc heme/lipid microsphere solution to rabbits demonstrates that it transports oxygen even in vivo and that it is cleared from the blood stream with a half-life time of approx. 1 h.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Triglyceride microsphere emulsified with 5,10,15,20-tetrakis[alpha,alpha,alpha,alpha-o-[2,2-dimethyl-20-[2-(trimethylammonioethoxy)phosphonatoxy]eicosanamido]phenyl] porphyrinatoiron (lipid-heme) provides a new totally synthetic oxygen-carrier (lipid-heme/microsphere) under physiological conditions . Oxygen-binding property of the lipid-heme/microsphere is comparable with that of red blood cell.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Double-sided porphyrinatoiron(II) bearing covalently bound axial imidazole, 5,10,15-tris(2,6-bis(3,3-dimethylbutyryloxy)phenyl)-20-(2-(3,3-dimethylbutyryloxy)-6-(5-imidazolylvaleroyloxy)phenyl)porphyrinatoiron (1b), was synthesized: The substituents of 1b are bound with the porphyrin only through ester bonds. 1b formed a stable O2 adduct reversibly in toluene at 25-degrees-C or with a lipid surfactant under physiological conditions.

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本人工臓器学会  

In vivo oxygen transporting capacity of totally synthetic artificial red cell (poly-liposome embedded lipid-heme) solution, which can bind and release molecular oxygen under physiological condition, was described. Exchange transfusion with the lipid-heme solution to six dogs weighing about 8kg was carried out. After the transfusion with 30ml/kg of the lipid-heme solution, the oxygen partial pressure of mixed venous blood increased from 30 to 50mmHg. Oxygen consumption by the lipid-heme was calculated to be 15ml/min (20% of total oxygen transport), which is corresponding to the lipid-heme concentration in the blood stream. Circulation half-life time in the blood stream was about 11hr. for the lipid-heme and about 26hr. for the liposome. Oxidation rate of the lipid-heme (met-formation) in the blood stream was slower than that of in vitro: only 6% of the lipid-heme was oxidized at 11hr. after the injection.

DOI： 10.11392/jsao1972.21.299

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Metal-ligand bonding properties of double-sided porphyrinato-iron(II) and -cobalt(II) complexes have been characterized by ESR, IR, and Mossbauer spectroscopy. The smaller A(N) value for a 1-methylimidazole (mim) adduct of 5,10,15,20-tetra(2,6-dipivaloyloxyphenyl)porphyrinatocobalt(II) compared to that of 5,10,15,20-tetra(2,6-di-tert-butylacetoxyphenyl)porphyrinatocobalt(II) suggested that the cobalt-imidazole bond is weak. The ESR spectrum of the dioxygenated double-sided series in fluid toluene indicated that an electrostatic interaction between the bound dioxygen and the ester fences was rarely found. The relaxation of steric strain on the rear side of the ring plane for axial imidazole bonding resulted in a lowering of the bound CO and O2 stretching frequencies of iron(II) complexes. This indicates that the pi-back donation from the d-pi orbital of the iron to the pi* orbital of the bound gaseous ligand could be controlled by the strength of the iron-imidazole bonding, which is regulated by the structure of the rear pocket on the macrocycle. The co-ordination structure of various ligands in double-sided porphyrinatoiron complexes is also discussed by means of Mossbauer parameters.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The kinetics of binding of O2 and CO to double-sided porphyrinatoiron(II) complexes having ester pockets on both sides of the porphyrin plane has been studied. The specific environment created by the four ester groups around the central iron(II) ion of 5,10,15,20-tetrakis(2,6-di-tert-butylacetoxyphenyl)-porphyrinatoiron(II) 2 results in binding which is not affected by solvation. The lower binding affinity for CO of 5,10,15,20-tetrakis(2,6-dipivaloyloxyphenyl)porphyrinatoiron(II) 1 compared to that of 5,10,15,20-tetra (o-pivalamidophenyl)porphyrinatoiron(II) is attributed to the unfavourable steric repulsions between the axial imidazole ligand and the pivaloyloxy groups, and is reflected in decreased association and increased dissociation rates. On the other hand, axial base ligation to 2 is not inhibited by the tert-butylacetoxy groups. Therefore, the lower binding affinity for O2 exhibited by 2 compared to that of an amide fenced porphyrin complex is ascribed to the loss of local polarity in the cavity. The less-polar ester groups of the double-sided porphyrinatoiron complex result in an increased rate of dissociation of O2. The activation energy for gaseous ligand association to complex 2 was determined.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

The synthesis and characteristic ligation behaviors of new ''double-sided porphyrinatoiron(II) complexes'' having two pockets of different spacing-size on each side of a ring plane and a larger sized one on both sides of a macrocycle, are described. The equilibrium constants for the imidazole derivatives to the four-coordinated porphyrinatoiron(II)s were increased in proportion to the size of the pocket-space, which is reflected by the DELTA-H term. The five-coordinated complexes of the porphyrinatoirons, obtained by the addition of 1,2-dimethyl-imidazole (1,2-dmin) in a toluene solution, formed stable and reversible dioxygen adducts at 25-degrees-C. The oxygen affinities for the iron(II) complexes increased as the steric bulk on the rear side of the porphyrin plane was relieved. These changes in the oxygen affinity are attributed to the strength of the pi-electron donation from the imidazole to the central iron ion.

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Web of Science

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Web of Science

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Responsible for pages：p. 82-88   Language：Japanese   Book type：Scholarly book

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Responsible for pages：p. 532-539   Language：English   Book type：Scholarly book

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Responsible for pages：p. 279-286   Language：Japanese   Book type：Scholarly book

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Responsible for pages：p. 1333-1336   Language：Japanese   Book type：Scholarly book

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Language：Japanese   Publisher：日本血液代替物学会  

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Language：Japanese   Publisher：日本脳循環代謝学会  

J-GLOBAL

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Language：Japanese   Publisher：日本血液代替物学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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