To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.

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A low molecular mass cysteine proteinase inhibitor was found and isolated from bovine hoof. Purification of the inhibitor to homogeneity was performed by carboxymethyl-papain-sepharose affinity and DE-52 ion exchange chromatographies. From 15 g of bovine hoof, 0.25 mg of cysteine proteinase inhibitor was isolated. It had 100 amino acid residues and molecular mass of 11 406 Da, and its N-terminus was blocked. The amino acid sequence of the inhibitor demonstrated high similarity with that of the family 1 group, especially bovine thymus-derived cystatin. Thus, in summary, bovine hoof contains a cystatin-type cysteine proteinase inhibitor, presumably a family 1 cystatin.

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