PROTEOLYSIS OF BOVINE BETA-CASEIN BY A PLASMIN-SEPHAROSE CONJUGATE

A plasmin-Sepharose conjugate (5.7 mg of active plasmin/g gel) was pre pared by covalent immobilization of bovine plasmin (EC 3.4.21.7) on ac tivated CH-Sepharose 4B, with formation of amide bonds between primary amino groups of plasmin and carboxyl-activated functions anchored to the gel. A strong decrease (60%) of the proteolytic activity of plasmi n for a chromogenic substrate was observed during dialysis performed b efore immobilization. However, a high binding yield and a good preserv ation of the proteolytic activity of immobilized enzyme (95%) were the n observed. The reactivity of free and immobilized plasmin with bovine beta-casein (beta-CN) were compared. Kinetic study of beta-CN proteol ysis indicated a perceptible loss of enzyme activity in plasmin-Sephar ose conjugate compared to free plasmin (V(max) = 3.8 10(-2) and 53.5 1 0(-2) OD unit/min, K(m) = 1.2 10(-4) et 6.2 10(-4) mol/l, respectively ). Electrophoretic study of the reaction mixtures showed that the prot eolysis of beta-CN f(1-105/7) by immobilized or free plasmin, producin g beta-CN f(29-105/7), succeeds the reaction of the whole beta-CN. Mic roparticle-enhanced nephelometric immunoassay of residual beta-CN, per formed in reaction supernatants, indicated there is no significant inf luence of beta-CN fragments (beta-CN f(1-105/7) and mixture of C-termi nal peptides beta-CN (29-209), (106-209), (108-209)) on the proteolysi s of the whole beta-CN. As already observed for other proteases, an al teration of the enzyme charge during the immobilization process might be the cause of the slight modification of the catalytic activity obse rved in plasmin-conjugate.