The amino acid sequence of the major alpha globin of the rat (Wistar, Rattus norvegicus)

Abstract

1. Rat (Wistar, Rattus norvegicus) was found to possess six distinctive haemoglobin components. The major haemoglobin component E was prepared by chromatography on column of DEAE-Sephadex using a pH gradient. Globin chains were separated on a CM-cellulose column which was eluted with increasing ionic strength of sodium ions in the presence of 8 M urea.

2. The amino acid sequence of the ∝ chain was substantially determined. The soluble tryptic peptides prepared from the native ∝ globin and from the aminoethylated ∝ globin were separated by peptide mapping. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble core peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human ∝ chain.

3. The amino acid sequence obtained in this study was compared with an amino acid sequence which was partially determined by an automated sequencer and partially by the homology method (Garrick et al. 1975). Disagreements between the two sequences were discussed and the favoured amino acid sequence for the rat major ∝ chain was proposed.

4. Residue 13(All) ∝ of rat is a cysteine, which is unique among the mammalian ∝ globins. The occurrence of this externally situated cysteine was suggested to be responsible for the in vitro crystallizing property of rat haemoglobins.

5. Structural and functional constraints of the haemoglobin molecule were shown to be a major selective force in the evolution of the rat ∝ chain.

6. A phylogenetic tree of the ∝ globins of rat, mouse, dog and rabbit was built by reconstructing their ancestral sequences. From this the time of divergence of rat (Rattus) and mouse (Mus) was calculated to be approximately 52-58 million years ago.