Axolotl hemoglobin: cDNA-derived amino acid sequences of two α globins and a β globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct α type globin polypeptides (αM and αm) and a common β globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for αM globin, 143 for αm globin and 146 for β globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established α type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that αM, αm, β and the previously known α major globin were adult types of globins and the other known α globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.