Structural heterogeneity of human hemoglobin A due to nonenzymatic glycosylation.

Human hemolysate contains several glycosylated minor hemoglobms (Hbs AI,~, A Ia2, Am, and AI,) which can be chromatographically separated from the major component, Hb Ao. The glucosyl-ketoamine linkage in Hb AI, can be detected calorimetrically by the thiobarbituric acid test. When chromatographed on Bio-Rex 70 resin, Hb A0 is eluted as a single peak following Hb AI,. We have found that the leading edge of Hb Ao, as well as Hb AI,, contains carbohydrate as detected by the thiobarbituric acid test. Both glycosylated components were comparably increased in the diabetic. There was a corresponding increase in the incorporation of tritium from [3H]borohydride into diabetic Hb A+ After Hb A0 was incubated with [‘4C]glucose it was chromatographed on Bio-Rex 70 resin. The specific activity profile corresponded closely to the thiobarbituric acid test profile. Parallel incubation with glucose having 3H bound to the second carbon atom confirmed that both the synthetic Hb AI, and the glycosylated Hb Ao have undergone the Amadori rearrangement to the more stable ketoamine linkage. We estimate that 8 to 10% of Hb A0 in normal red cells is glycosylated. Autoradiograms of tryptic peptide maps indicate that several sites on both the a and /3 chains are modified, including the NH2 terminus of the (Y chain. Comparison of ion exchange liquid chromatograms of synthetic lysino-ldeoxysorbitol with those of acid hydrolysates of [3H]borohydride-reduced native Hb A0 and [‘4C]glucosyl Hb A0 shows that the glucose is bound to lysines. This nonspecific reaction of glucose with lysine probably occurs in other proteins and may contribute to some of the long term complications of diabetes.