Structural Analysis of the Minor Human Hemoglobin Components: Hb A_Ia1, Hb A_Ia2 and Hb A_Ib

Human hemolysate contains several minor hemoglobin components, including Hb A_Ia1, Hb A_Ia2, Hb A_Ib and Hb A_Ic which are post‐translational modifications of the major component, Hb A_o. Hb A_Ic is known to contain glucose attached to the N terminus of the β chains by a ketoamine linkage. We separated the α and β globin chains from purified Hb _Ia1, Hb A_Ia2 and Hb A _Ib by ion‐exchange chromatography. The β 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 β A_Ia1 chain and 1 phosphate residue with each β A_Ia2 chain. Hb A_Ia1, Hb A_Ia2 and Hb A_Ib were all found to be contaminated by non‐globin proteins. Protein‐sequencing approaches demonstrated that the N termini of β A_Ia1, β A_Ia2 and β A_Ib were blocked. In support of this conclusion, analysis of tryptic digests of β A_Ia2 and β A_Ib revealed modified N‐terminal peptides. We conclude that, like Hb A_Ic, components Hb A_Ia1, Hb A_Ia2 and Hb A_Ib also contain a sugar moiety linked to the N terminus of the β chain.