Quantitative Characterization of Bovine Plasminogen Binding to Caseins

Abstract

The binding of bovine plasminogen to whole casein, α s-casein, β-casein, and κ-casein is responsible for the progressive proteolysis of milk and dairy products. A sensitive and accurate microparticle-enhanced nephelometric immunoassay was developed to measure free plasminogen after interaction between bovine plasminogen and caseins and the quantitative parameters of plasminogen/casein binding were established. Two classes of binding sites for plasminogen were found in this study on each of the investigated caseins. Their dissociation constants ( K d ) were determined by varying the plasminogen concentration at pH 6.6 and performing Scatchard analysis. The two binding sites appeared to be one of high affinity ( K d = 32 nM) and the other of lower affinity ( K d > 370 nM). The number of both binding sites per casein monomer was low (0.04 to 0.53). The great propensity of casein monomers to self-associate in homopolymers where plasminogen binding sites could be hidden and in copolymers present in bovine milk in the form of whole casein micelles accounts for calculated binding sites <1 per monomer.