Purification and properties of an acidic protein from rat skin.
An acidic protein, extractable in neutral salt solutions from rat skin, was markedly enriched when precipitated by dialysis against 0.5 M acetic acid. After dissolving the precipitate in 0.5 M Tris-HCl buffer, pH 8.0, the protein was disaggregated by the addition of the nonionic detergent Triton X-100 and purified by chromatography on Sephadex G-100 and DEAE-Sephadex A-50 columns. The protein isolated under nondenaturing conditions appeared to be essentially homogeneous by its migration as a single band on (a) cellulose acetate membrane electrophoresis at pH 8.6; (B) 4% and 7.5% polyacrylamide gel electrophoresis at ph 8.9; (C) sodium dodecyl sulfate (10%) polyacrylamide gel electrophoresis at pH 7.0; and by (d) its complete freedom from collagen, the major contaminating protein. The molecular weight of the protein was determined as 76,000 +/- 2,000 from its electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels and 75,000 from its elution volume in Sephadex G-100 columns. Reduction and alkylation of the protein failed to generate smaller subunits. The amino acid composition of the protein showed that it was relatively rich in glutamic and aspartic acids, which together comprised 25% of its total residues. Hydrophobic amino acids like phenylalanine, leucine, isoleucine, valine, methionine, alanine, proline, and cystine accounted for about 34% of the total residues in the protein. No free NH2-terminal amino acid could be detected in the purified protein by the dansylation method. Each mole of protein contained 11 mol of phosphate. Triton X-100 was necessary for achieving nondestructive disaggregation of the acidic protein. Each mole of protein bound about 3200 mol of Triton X-100 or 10 mol of Congo red. While the detergent binding could be reversed by dialysis, Congo red formed a stable complex with the protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D003224:Congo Red; D004588:Electrophoresis, Cellulose Acetate; D000882:Haplorhini; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000032:analysis; D011092:Polyethylene Glycols; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D012867:Skin / Q000032:analysis*
D000596:Amino Acids; D009943:Organophosphorus Compounds; D011506:Proteins; D003224:Congo Red; D011092:Polyethylene Glycols