Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2 (HbD2). Their pI values are 7.53 ± 0.02, 7.37 ± 0.02, 6.92 ± 0.04 and 6.72 ± 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 are identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weights and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin or of aggregation due to overloading or formation of multiple units of hemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.
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