Auto-Oxidation of Human Hemoglobin and the Roles of Distal Heme Pocket Substitutions

Abstract

This study investigates the auto-oxidation reaction of human normal adult hemoglobin (Hb A) and the effects of distal heme pocket substitutions, P50, and tetramer-dimer dissociation on the rate and mechanism of auto-oxidation. Recombinant hemoglobins (rHbs) with single amino acid substitutions at helical positions E11 and B10 have been expressed in Escherichia coli and purified, as well as di-α linked and octameric rHbs. These rHbs include: rHb (αV62L), rHb (αV62I), rHb (βV67L), rHb (βV67I), rHb(αL29W), rHb(αL29F), rHb(α-Gly-α/β gene di-α/β), rHb(diαL29F), rHb(diαL29W), and rHb(βG83C). Auto-oxidation measurements were conducted with 32μM heme in MES buffer (pH 6.5) for 60 hours at 35 °C. A monophasic nature of auto-oxidation has been observed for Hb A and a biphasic nature for all other rHbs. In comparison to the other mutants, including di-α linked and octameric rHbs, rHb (αL29F) is most resistant to oxidation and rHb (αL29W) is the least resistant to oxidation. Characterization of three novel rHbs; (βL28F, βV67I), (αL29F, βV67I), (αL29F,αV62I) will test whether the fast and slow phases of the observed biphasic nature of auto-oxidation can be attributed to the mutated and wild-type subunits, respectively. Our studies will also provide new insights into the roles of amino acid residues in the distal heme pockets on the structure-function relationship in hemoglobin(Supported by NIH grants HL-024525 and GM-084614, HHMI, and The Arnold and Mabelle Beckman Undergraduate Research Scholars Program).