The rennet-induced clotting of para-kappa-casein revisited: inhibition experiments with pepstatin A

The proteolysis of micellar kappa-casein by rennet was followed by SDS-polyacrylamide gel-electrophoresis and the clotting of the para-kappa-casein formed by absorbance measurements. Up to a degree of proteolysis of about 0.4 the enzyme-inhibitor pepstatin A proved able to instantaneously stop the clotting. This effect is explained by the rapid condensation of monofunctional, monomeric and polymeric particles of para-kappa-casein. At higher degrees of proteolysis pepstatin was no longer able to completely block the polymerization. This is explained by the retardation of the condensation of the monofunctionals as their size grows larger. A kinetic analysis of the enzyme-controlled stage of the clotting process predicts that the system should gel at an early degree of proteolysis of about 0.07. The actual gel points occur at considerably higher degrees of proteolysis. This suggests that the enzymic attack of the polymeric inert kappa-casein particles is not completely at random. Primary micelles of kappa-casein, however, are degraded by random attack rather than by a 'catch-and-razor' mechanism.