Tetrameric hemoglobins (Hbs) A and D were isolated from red blood cells of the Aldabra giant tortoises, Geochelone gigantea, by a hydrophobic interaction chromatography. After reduction and S-pyridylethylation, two sets of two types of α-chains (α-1 and α-2) and one β-chain were purified from the major Hb A and minor Hb D in molar ratios of about 1:1:2, respectively, by a reversed-phase column chromatography. The complete amino acid sequences of the three globin-chains from Hb A were determined: 141 amino acid residues for the two α-chains and 146 amino acid residues for the β-chain. Using computer analysis (amino acid maximum homology), the two α-chains shared a 96.5% sequence identity and had low sequence identities (37.8% for α-1 and 35.8% for α-2) with the β-chain of the same species, G. gigantea.

We constructed a phylogenetic tree of 28 primary globin structures from Reptilia (7 species of squamates, 4 species of turtles, 3 species of crocodiles and 1 species of sphenodontids), including the three globins of G. gigantea Hb A. The following results were obtained: (1) The two terrestrial species of Geochelone (G. gigantea and G. carbonaria) were closely related: 139 amino acid residues (95.2%) of the two β-globin chains were conserved; (2) Based on the divergence patterns of globin-chains, the sea turtle Caretta caretta was shown to be unusual relatedness form the groups of terrestrial and freshwater species in turtles. The molecular relationships appearing on the phylogenetic tree also support the traditional classification of reptiles and partly confirm previous molecular studies of reptilian hemoglobin evolution.