Inhibition of Plasmin by β-Lactoglobulin Using Casein and a Synthetic Substrate

Abstract

Bovine plasmin (EC 3.4.21.7) activity was measured on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide and acid casein in the presence of native and heat-denatured β-lactoglobulin (denatured at 100°C for 15 min before being mixed with plasmin solutions). Native or denatured β-lactoglobulin was then heated with plasmin at 60°C for 15 min. Enzyme activity again was estimated after this mild heat treatment. Native and denatured β-lactoglobulin inhibited the action of plasmin on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide and casein. The mild heat treatment (60°C for 15 min) caused stronger inhibition of the activity of plasmin against casein and the synthetic substrate. For H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide, inhibition was competitive in unheated mixtures, but heating β-lactoglobulin with plasmin changed inhibition type to mixed. This change suggests a heat-dependent interaction between plasmin and β-lactoglobulin. Native β-lactoglobulin was more inhibitory of plasmin's action against casein than was denatured β-lactoglobulin. The converse was observed when plasmin activity was measured with the synthetic substrate.