Tetrakis{(R,R,R,R-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 °C) in the same manner as hemoglobin and
myoglobin. The O2-adduct complex had a remarkably long lifetime (ô1/2: 5 h). The O2-binding affinity
[P1/2
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.