Structure and Function of the Isolated Hemoglobins of the American Eel, Anguilla rostrata

Abstract

Abstract Two electrophoretic hemoglobin patterns occur in hemolysates from the American eel, Anguilla rostrata. Type I hemolysates have two major components, I-F and I-S. Type II hemolysates have three major components, II-F, II-M, and II-S. Each kind of hemolysate also has at least one minor electrophoretic component. Amino acid analysis and tryptic peptide patterns of the isolated polypeptide chains indicate that no polypeptide chains are common to the S components and to either the F or the M component. The β chains of the three major components of the type II hemolysate are all different; the II-F β and II-M β chains are similar but differ by at least one peptide. Both differ greatly from the II-S β chain. The α chains of Components II-F and II-M appear either to be identical or very similar and to differ greatly from the α chain of II-S. These results are most compatible with the assumption that the β chains of the anodal Components F and M are controlled by two nonallelic loci. The globin of Component I-F yielded two α chains, one of which appeared identical or very similar to the α chains of II-F and II-M, whereas the other I-F α chain differed by at least one peptide. The oxygen equilibria of Components II-F and II-M appear to be identical. In the absence of phosphate they are characterized by a very large Bohr effect below pH 7: Δ log P50/Δ pH ≅ -1.6 in the pH interval, 6.5 to 6.8. The Hill coefficient, n, decreases from 1.3 to 1.4 above pH 7 to about 0.9 near pH 6.5. The oxygenation properties of Component II-S are entirely different from those of Components F or M. A substantial reversed Bohr effect is present in the absence of phosphate between pH 6.8 and 8.4; Δlog P50/ΔpH = +0.3. Addition of 6 moles of ATP per mole of hemoglobin (tetramer) causes a large pH-dependent decrease in oxygen affinity and a substantial rise in apparent cooperativity. The Hill coefficient, n, rises from 1.6 to 1.8 to about 2.4 to 2.8 depending on the pH. The magnitude of the Bohr effect decreases to close to zero upon ATP addition. Carbon dioxide increases the reversed Bohr effect in the absence of phosphate: Δlog P50/ΔpH becomes +0.75. The COOH-terminal histidyl residue of the β chains of mammalian hemoglobin is believed to play a decisive role in the mechanism of the Bohr effect (Perutz, M. F. (1970) Nature 228, 726–739). Component F has the COOH-terminal sequence -Tyr-His, but the corresponding sequence in Component S is -Gln-Tyr-Phe This difference may largely account for the different behavior of the two hemoglobins. The Component S β chain has only 2 histidyl residues, whereas all other eel hemoglobin chains examined have 5 to 6 histidines per chain.