Physicochemical characterization of cross-linked human serum albumin dimer and its synthetic heme hybrid as an oxygen carrier

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°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 O ₂ under physiological conditions (37°C, 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.