14C-Methylated beta-casein as a substrate for plasmin, and its application to the study of milk protein transformations.

A method was investigated for the sensitive radiochemical assay of milk protein transformations. beta-Casein was reductively methylated with NaBH4 and H14CHO giving a product of spec. act. 4.42 microCi/mg in which a maximum of 12% of the lysine residues were labelled. Methylation did not alter the electrophoretic or chromatographic properties of the protein, or its pattern of proteolysis by plasmin. The substrate susceptibility of 14C-methylated beta-casein towards plasmin was determined by measuring radioactivity transfer to the proteolytic fragments gamma 2- and gamma 3-casein. Compared with the native protein, no decrease in substrate susceptibility was detected. The presence in milk of the plasmin-like enzyme, milk proteinase, was demonstrated and its activity at 4 degrees C was quantified by examination of the fragmentation pattern of added 14C-methylated beta-casein. It was concluded that 14C-methylated protein substrates, prepared by reductive methylation, are well suited to the study of hydrolytic enzymes and that they can provide valuable information on milk protein transformations. In particular, no interference was encountered with the rate of cleavage by plasmin when the level of methylation was kept to a minimum.