Human serum albumin bearing covalently attached iron(II) porphyrins as O2-coordination sites

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 O2 reversibly under physiological conditions (pH 7.3, 37 degrees C) in the same manner as hemoglobin and myoglobin. The O2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O2-binding affinity [P(1/2)O2: 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.