Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.