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Steroid Binding Properties of Some Peptide Fragments of Bovine Serum Albumin Obtained on Peptic Digestion

1972; Elsevier BV; Volume: 247; Issue: 24 Linguagem: Inglês

10.1016/s0021-9258(20)81812-2

1083-351X

William H. Pearlman, I.F.F. Fong,

Hormonal and reproductive studies

Abstract To elucidate the structural characteristics of bovine serum albumin (BSA) which allow for steroid binding, BSA was digested with pepsin under conditions which afford maximal retention of steroid binding activity consistent with a maximal degree of peptide fragmentation. Digestion with pepsinBSA (w/w), 1:3000, pH 3.0, for 2 hours at 25° afforded a complex mixture of peptide fragments with molecular weights of about 14,000 (a major class of components), and 27,000 and 34,000 (minor classes) estimated on sodium dodecyl sulfate gel electrophoresis of the digest. As much as 40 to 50% of the initial steroid binding activity was retained in the BSA digest; the steroids studied were progesterone, testosterone, and 17β-estradiol. Steroid binding activity was empirically defined as the product of the ratio of bound to unbound steroid times the reciprocal of the total protein or peptide concentration (gram per liter) in an assay system containing Sephadex G-25 or G-10 under conditions of equilibrium. The crude BSA digest was treated successively with 2% and 10% trichloroacetic acid; the respective trichloroacetic acid-precipitable peptides were chromatographed on Sephadex G-75. The 2% trichloroacetic acid chromatographic fractions exhibited a relatively high progesterone binding activity, and also appreciable testosterone and estradiol binding, whereas successive chromatographic fractions of the 10% trichloroacetic acid fraction exhibited diminishing steroid binding activity. Binding activity correlated with the absorbance ratio, 280:258 nm, of the respective chromatographic fractions, suggesting that peptide fragments which are richer in tyrosine tend to retain steroid binding activity to a greater degree. Although the bulk of the peptide material remains to be resolved, two peptide fragments, KL and VI, were isolated from the 2% trichloroacetic acid and 10% trichloroacetic acid fractions, respectively. Peptide KL (mol wt 10,050) is rich in tyrosine. The equilibrium constant, nk, for the formation of a complex of peptide KL with progesterone, testosterone, or 17β-estradiol at 25° was about 0.44, 0.18, or 0.33 x 104 m-1, respectively. Peptide VI (mol wt 2766) appears to be identical with the NH2-terminal or Asp fragment of BSA previously isolated by Peters and Hawn ((1967) J. Biol. Chem. 242, 1566). Peptide VI contains no tyrosine and exhibits very little steroidbinding affinity.