Glycosylation of hemoglobin in vitro: affinity labeling of hemoglobin by glucose-6-phosphate.

Abstract

About 5% of Hb in normal human red cells is glycosylated. This minor component, designated Hb Alc, is increased at least 2-fold in patients with diabetes mellitus. To determine the mechanism for the formation of Hb Alc in vivo, human Hb was incubated with glucose and metabolites of glucose. [14C] G-6-P reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of CO or 2,3-diphosphoglycerate (2,3-DPG). Purified G-6-P Hb had a lowered O2 affinity and decreased reactivity with 2,3-DPG compared to Hb A. G-6-P behaved as a 2,3-DPG analog and reacted specifically at the NH2-terminal amino group of the .beta. chain. In contrast, the interaction of Hb with glucose was much slower, and was unaffected by CO or 2,3-DPG. Neither glucose-1-phosphate, fructose-1-phosphate, fructose-6-phosphate nor fructose-1,6-diphosphate formed a reaction product with Hb. G-6-P behaves as an affinity label with the phosphate group forming electrostatic bonds at the 2,3-DPG binding site and the aldehyde group reacting with the NH2-terminal amino group of the .beta. chain. G-6-P Hb may be an intermediate in the conversion of Hb A to Hb Alc.