Proteolysis of bovine β-casein by a plasmin-Sepharose conjugate

Abstract

Summary -A plasmin-Sepharose conjugate (5.7 mg of active plasmin/g gel) was prepared by covalent immobilization of bovine plasmin (EC 3.4.21.7) on activated CH-Sepharose 48, 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 plasmin for a chromogenic substrate was observed during dialysis performed before immobilization. However, a high binding yield and a goOO preservation of the proteolytic activity of immobilized enzyme (95%) were then observed. The reactivity of free and immobilized plasmin with bovine p-easein (P-CN) were compared. Kinetic study of p-CN proteolysis indicated a perceptible loss of enzyme activity in plasmin-Sepharose conjugate compared to free plasmin (Vmax = 3.8 10-2 and 53.5 10-2 00 unit/min, Km = 1.2 1Q-4 et 6.2 10-4 molli, respectively). Electrophoretic study of the reaction mixtures showed that the proteolysis of p-CN f(1-10517) by immobilized or free plasmin, producing p-CN f(29-1 0517), succeeds the reaction of the whole p-CN.Microparticle-enhanced nephelometric immunoassay of residual p-CN, performed in reaction supernatants, indicated there is no significant influence of p-CN fragments (P-CN f(1-1 0517) and mixture of C-terminal peptides p-CN (29-209), (106-209), (108-209)) on the proteolysis of the whole p-CN. As already observed for other proteases, an alteration of the enzyme charge during the immobilization process might be the cause of the slight modification of the catalytic activity observed in plasmin-conjugate.