Structural heterogeneity of human hemoglobin A due to nonenzymatic glycosylation

Human hemolysate contains several glycosylated minor hemoglobins (Hbs A(Ia ₁), A(Ia ₂), A(Ib), and A(Ic)) which can be chromatographically separated from the major component Hb A ₀. The glucosyl-ketoamine linkage in Hb A(Ic) can be detected colorimetrically by the thiobarbituric acid test. When chromatographed on Bio-Rex 70 resin, Hb A ₀ is eluted as a single peak following Hb A(Ic). We have found that the leading edge of Hb A ₀, as well as Hb A(Ic), 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 ₀. After Hb A ₀ was incubated with [ ¹⁴C]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 A(Ic) and the glycosylated Hb A ₀ have undergone the Amadori rearrangement to the more stable ketoamine linkage. We estimate that 8 to 10% of Hb A ₀ in normal red cells is glycosylated. Autoradiograms of tryptic peptide maps indicate that several sites on both the α and β chains are modified, including the NH ₂ terminus of the α chain. Comparison of ion exchange liquid chromatograms of synthetic lysino-1-deoxysorbitol with those of acid hydrolysates of [ ³H]-borohydride-reduced native Hb A ₀ and [ ¹⁴C]glucosyl Hb A ₀ 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.