The Structure of Sheep Hemoglobins

The existence of two adult hemoglobin types, designated as hemoglobin A (or hemoglobin II) and hemoglobin B (or hemoglobin I), in various breeds of sheep is well established (for reference see van Vliet and Huisman (1)). The two molecular species have been separated both by electrophoresis, with which hemoglobin A showed the highest mobility at alkaline pH, and by chromatography on either Amberlite IRC-50 (2) or carboxymethyl cellulose (3), on which hemoglobin B was more strongly adsorbed to the cation exchange agent. Hemoglobin B was found to be less soluble than hemoglobin A in concentrated phosphate solution at a pH 6.5 (2). Sedimentation coefficients of 4.3 were obtained at neutral pH for both species (4), indicating a molecular weight of approximately 66,000. Sedimentation velocity studies at a pH of 4.7 and at a pH of 10.5 and higher have shown that both types dissociate into subunits, and that the dissociation rate of hemoglobin B was slightly faster at both pH values (4). The subunits were also separated by chromatography with acid Amberlite IRC-50 with elution by a urea gradient, and by starch gel electrophoresis in a formic acid-formate buffer at pH 1.9 (5). On end group analyses of a sheep hemoglobin of unspecified type, two different NH*-terminal amino acid sequences were found, valylleucyl and methionylglycyl (6, 7). The data from all of these studies indicate that both sheep hemoglobins A and B are composed of two different kinds of polypeptide chains. In a previous paper (1)) we described the occurrence of a third hemoglobin type, hemoglobin C, which was occasionally observed in small quantities in sheep heterozygous for the hemoglobins A and B or homozygous for hemoglobin A. This third species became a major fraction during experimental anemia, resulting from loss of blood, through replacement of hemoglobin A, but not of hemoglobin B, in a sheep heterozygous for the two hemoglobin types, while the converse process was observed during the recovery phase of the anemia. Hemoglobin C can be distinguished from hemoglobin A and hemoglobin B: (a) electrophoretically, since hemoglobin C has the lowest mobility at pH 8.1; (b) by chromatography on DEAE-cellulose, because hemoglobin C is eluted as the first hemoglobin fraction; and (c) by solubility in concentrated salt solutions, as the solubility of hemoglobin C is intermediate between those of the hemoglobins A and B. Molecular hybridization of the three species with canine hemoglobin and starch gel electrophoretic studies of the isolated globins in formic-acid formate buffer, pH 1.9, indicated the pres-