Reductive hydroxyethylation of hemoglobin A. Functional properties of hemoglobin A selectively hydroxyethylated or dihydroxypropylated at the alpha-amino groups.

Abstract

The reactivity of the amino groups of hemoglobin A toward reductive hydroxyethylation with glycolaldehyde in the presence of NaCNBH3 has been investigated. The alpha-amino groups of the alpha- and beta-chains of hemoglobin A are the most reactive amino groups of the protein for reductive hydroxyethylation; both sites are about equally reactive. Similarly, on reaction of hemoglobin A with glyceraldehyde in the presence of NaCNBH3, the alpha-amino groups of both the alpha- and beta-chains of the protein undergo selective reductive dihydroxypropylation. Modification of the alpha-amino groups of hemoglobin A either by reductive hydroxyethylation or reductive dihydroxypropylation apparently lowers the pKa of the alpha-amino groups and results in an early elution of the modified protein from CM52 chromatography compared with that of the unmodified protein as well as protein modified at the epsilon-amino groups. By hybridization of the modified chains with unmodified chains, new derivatives of hemoglobin with dihydroxypropyl or hydroxyethyl moieties on Val-1 (alpha) or Val-1(beta) of the protein have been prepared for the functional studies. Modification of the alpha-amino group of Val-1(alpha) by reductive hydroxyethylation or by reductive dihydroxypropylation resulted in an increased oxygen affinity of hemoglobin A, whereas the same modification of the alpha-amino group of the beta-chain resulted in a slight decrease in the oxygen affinity. The Hill coefficient of all four derivatives was 2.7, nearly the same as that of the native protein, showing normal heme-heme interactions in these derivatives. Both 2,3-diphosphoglycerate and chloride modulate the oxygen affinity of these hybrids; the influence of 2,3-diphosphoglycerate on decreasing the oxygen affinity of the hybrids with alkyl groups at Val-1(beta) is relatively less than that of the native molecule. On the other hand, when the modification was on Val-1(alpha), the relative decrease in oxygen affinity of the hybrids upon binding of 2,3-diphosphoglycerate was of the same order as with the native protein. The chloride ion-mediated relative decrease in the oxygen affinity of the hybrids with alkyl groups at Val-1(beta) was nearly the same as that seen with the Val-1(alpha) hybrids, and lower than that with the native molecule. The relative influence of hydroxyethylation or of dihydroxypropylation at a given site was nearly the same, suggesting that the size of the alkyl chain has little to do with the observed changes in the oxygen affinity. The influence of reductive alkylation of the alpha-amino groups with these two hydroxyaldehydes probably reflects the change (decrease) in the pKa of the reductively hydroxyethylated or dihydroxypropylated alpha-amino group as compared with that of the unmodified alpha-amino groups.