title: Physicochemical characterization of cross-linked human serum albumin dimer and its synthetic heme hybrid as an oxygen carrier.
abstract: 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 (pI: 4.8) and denaturing temperature (65 degrees C). 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 125I-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 O2 under physiological conditions (37 degrees C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O2-recombination reaction. The O2-binding affinity and the O2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated.
authors.affiliation: Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Komatsu
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Oguro
authors.forename: Yuji
authors.initials: Y
authors.lastname: Teramura
authors.forename: Shinji
authors.initials: S
authors.lastname: Takeoka
authors.forename: Junpei
authors.initials: J
authors.lastname: Okai
authors.forename: Makoto
authors.initials: M
authors.lastname: Anraku
authors.forename: Masaki
authors.initials: M
authors.lastname: Otagiri
authors.forename: Eishun
authors.initials: E
authors.lastname: Tsuchida
mesh_terms: D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D019281:Dimerization; D006420:Hemeproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D009997:Osmotic Pressure; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012709:Serum Albumin / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D000075462:Serum Albumin, Human; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D006420:Hemeproteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; C400056:rHSA-FeP protein, human; D010100:Oxygen; D000075462:Serum Albumin, Human