Effect of Solvent Composition on the Precipitation of Cathepsin D Hydrolysates of α_s1-Casein with Trichloroacetic Acid

Abstract

　Practically pure α_s1-casein was separated from bovine skim milk by the precipitation procedure in 50% ethanol and urea solutions. It was hydrolysed by cathepsin D at 37℃ in 0.1 M sodium phosphate buffer (pH 6.0). A number of degradation products were observed by urea-polyacrylamide gel electrophoresis (PAGE) after the action of cathepsin D on α_s1-casein but these components as well as undegraded α_s1-casein were mostly precipitated by the addition of 2% (W/V) trichloroacetic acid (TCA). The addition of urea to the precipitation system partly released degradation products into the supernatant. In a precipitation system, 2% TCA-3.3 M urea, inclusion of ethylenediamine-tetraacetic acid (EDTA) up to 40 mM somewhat promoted precipitate formation. By adding Triton X-100 (0.1%) to the precipitation system, a considerable part of components released into the supernatant. This increase in solubility was further promoted by the addition of both Triton X-100 and EDTA. These facts suggest that hydrophobic interactions between peptides including undegraded protein must be taken into account in performing fractionation of enzymatic degradation products of α_s1-casein.