Studies on Monotreme Proteins. V. Amino Acid Sequence of the a-Chain of Haemoglobin From the Platypus, Ornithorhynchus anatinus

Abstract

Blood from the platypus contained three haemoglobins which were separated by chromatography on DEAE-Sephadex. The major component, Hb-I, was converted to globin and fractionated into the oc-and p-chains by chromatography on eM-cellulose in 8M urea-thiol buffers, and the complete amino acid sequence of the 141 residues of the oc-chain were determined. Peptides derived from the oc-chain by tryptic digestion were isolated by paper ionophoresis and chromatography. The amino acid sequences were determined by the dansyl-Edman procedure or by further digestion with other enzymes.The amino acid sequence showed 16 changes from the oc-globin of the other extant monotreme, the echidna. There were 39 changes from the human oc-globin sequence. By comparison with the 'contact sites' in horse haemoglobin there is one change in the oc-haem contacts, four changes in the OCI-PI contacts and no changes in the OC,-P2 contacts.The date of divergence of the monotremes from the other mammals was estimated to be 180± 37 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. The significance of this estimate in relation to the time of divergence of marsupials is discussed.

Fitting the Gene Lineage into its Species Lineage, a Parsimony Strategy Illustrated by Cladograms Constructed from Globin Sequences

“…[6][7][8][9] In this set of 35 alpha sequencies, two actual GDs are required, one for the two echidna chains (ECH IB and IIA) and another for the two chicken chains (CHI All and Al). These real GDs are desig- The ordinate scale in millions of years is inferred from fossil evidence on ancestral splitting times of the species from which the sequences come.…”

“…Since the duplication giving rise to these two chains is placed in the species lineage to echidna after this lineage separates from that to platypus (Figs. [6][7][8][9], there is no need to postulate any ensuing GE events.…”

Oxygen Binding of Monotreme Haemoglobins I. Oxygen Binding Parameters of the Haemoglobins of Platypus, Ornithorhynchus anatinus, and Echidna, Tachyglossus aculeatus

“…The substitutions at the oc 1 ,81 contacts and at the internal positions appear Table 3. Significant amino acid differences in echidna and platypus haemoglobins Sequence data are from Whittaker et al (1972Whittaker et al ( , 1973, and Whittaker and Thompson (1974). The functional significance is from Goodman et al (1975) to have sufficiently similar stereochemical properties that they are unlikely to have a significant effect.…”

“…In an effort to understand the phylogenetic relationships of the monotremes the amino acid sequences of the f3-chain (Whittaker et al 1972) and IX-chain (Whittaker et al 1973) of echidna haemoglobin IB were determined as well as the sequences of the IX-chain (Whittaker and Thompson 1974) and f3-chain (Whittaker and Thompson 1975) of platypus haemoglobin. Calculations based on the differences in amino acid sequence of the IX-chain suggested that monotremes diverged from other mammalian groups 211 million years ago, while calculations based on the f3-chain gave 132 million years for the divergence date.…”

Myoglobin of the Shark Heterodontus Portusjacksoni: Isolation and Amino Acid Sequence

“…Citraconylation followed by tryptic digestion and fractionation on DEAE-cellulose was done as previously described (Whittaker and Thompson 1974) using 200 mg of carboxymethylated myoglobin. Samples of each fraction (25,u1 made to 0·5 ml with water) were taken for radioactive counting with 5 ml of the mixture of Butler (1961).…”