Demonstration of a minor hemoglobin with modified alpha chains and additional modified hemoglobins in normal and...

We have attempted to separate various hemoglobins in 10 normal and 11 diabetic persons by Bio-Rex 70 chromatography at 4 degrees C with an exponentially increasing sodium phosphate gradient. Minor hemoglobins, namely AIa1, AIa2, AIb1, AIb2, AIb3, AIc, AId1, AId2 and AId3, have been separated and eluted in that order. Most of these minor hemoglobins were newly observed and could not be detected by the previous chromatographic techniques. On the basis of colorimetric assay protein-bound ketoamine was present in all the minor hemoglobins, which was confirmed by chromatographic separation of hemoglobins after reduction with NaB3H4. All the minor hemoglobins, with the exception of Hb AIa1, HbIa2, and Hb AIb1, showed a 2-fold increase in the diabetic patients. Hb AIc (5.0% in normals; 9.0% in diabetics) and Hb AId3 (1.9% in normals; 3.2% in diabetics) were present in the largest amounts. Both Hb AId3 and Hb Ao showed a decrease in oxygen affinity in the presence of 2,3-diphosphoglycerate, whereas Hb AIc showed no effect. Separation of globin chains by cellulose acetate electrophoresis at pH 8.6 showed that the cathodal mobility of the alpha chains of Hb AId3 was slower than those of Hb Ao. Glycosylation of Hb with [14C]glucose followed by separation of hemoglobins by two Bio-Rex 70 chromatographic methods indicated that the minor Hb formed by glycosylation of the alpha-chain amino-terminus was separated from Hb Ao, while the minor hemoglobins formed by the glycosylation of epsilon-NH2 groups chromatographed with Hb Ao.