A method for determination of γ-casein and its use for investigating proteolysis in bovine milk

Abstract

SummaryA convenient and sensitive method for determining γ-casein content of skim milk is described. It is based on quantitative separation in which the soluble fraction obtained from skim milk (1·5 ml) in a solvent system consisting of 50% (v/v) ethanol, 0·4 M-Na thiocyanate and 0·15 M-CaCl2 was applied to a small column of DEAE-cellulose and eluted with 0·02 M-Tris-HCl buffer (pH 8·0) containing 0·03 M-NaCl and 6 M-urea. Protein in this fraction (5 ml) was measured from its absorbance at 280 nm. The method is applicable also to heated skim milk. Its availability for investigating proteolysis in milk is expected from the fact that incubation of skim milk with porcine plasmin at 37 °C for 3 h increased γ-casein content, an increase that was lowered by addition of soyabean trypsin inhibitor. Incubation of raw skim milk with 0·02% (w/v) NaN3 resulted in increase in γ-casein with time and with increasing temperature. This was accompanied by a time lag, the length of which increased with decreasing temperature. The degree of proteolysis in skim milk, expressed as the amount of increase in γ-casein after 20 h of incubation at 37 °C, showed two pH optima, one at pH 8·0 and the other at pH 7·2–7·5. These results suggested that the present method.of γ-casein determination can be used for more precise studies of the milk plasmin system in which important factors, such as plasminogen activator and inhibitors, are involved.