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Isolation, Amino Acid Sequence and Copper(II)-binding Properties of Peptide (1–24) of Dog Serum Albumin

1974; Elsevier BV; Volume: 249; Issue: 18 Linguagem: Inglês

10.1016/s0021-9258(20)79899-6

1083-351X

Joan W. Dixon, Bibudhendra Sarkar,

Analytical Chemistry and Chromatography

Abstract The NH2-terminal peptide fragment (1–24) of dog serum albumin was obtained by a limited peptic hydrolysis of whole albumin. The peptide fragment was isolated and subsequently purified to homogeneity by trichloroacetic acid fractionation, Sephadex gel filtration, and high voltage electrophoresis at pH 2.0. The purity of the peptide was established by 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) analysis of the NH2-terminal residue, by amino acid analysis and by cellulose acetate electrophoresis at pH 8.6. The complete sequence, obtained by dansyl-Edman degradation on the whole peptide and by characterization of its tryptic peptides, was shown to be Glu-Ala-Tyr-Lys-Ser-Glu-Ile-Ala-His-Arg-Tyr-Asn-Asp-Leu-Gly-Glu-Glu-His-Phe-Arg-Gly-Leu-Val-Leu, which gave a molecular weight of 2847. The amide charge was obtained by amino acid analysis of residues released after enzymic digestion of the tryptic peptide. It was proposed earlier that in dog serum albumin the lack of specificity of Cu(II) binding was due to the absence of a histidine residue in position 3 (Appleton, D. W., and Sarkar, B. (1971) J. Biol. Chem. 246, 5040–5046). The present results show that a single base change in the albumin gene has replaced the important histidine-3 residue, critical to the binding of Cu(II), with a tyrosine residue. The homology between the NH2-terminal peptide from dog serum albumin and those from bovine, rat and human is discussed. To elucidate the role of these first 24 residues in the observed binding phenomenon of the intact dog serum albumin molecule, Cu(II)-binding properties of the peptide fragment were investigated by spectrophotometric and proton displacement studies. In both the intact protein and the 1–24 peptide fragment, the first equivalent of Cu(II) is distributed between at least two sites, indicating a nonspecific binding. This partitioning of the first equivalent of Cu(II) probably occurs between the NH2-terminal binding site and another site containing a histidine residue. Thus, a mutation in the codon specifying residue 3 in the dog peptide has resulted in an altered Cu(II) binding site of reduced specificity.