Casein Interference in Bovine Plasmin Assays Using a Synthetic Substrate

¹ Department of Nutrition and Food Sciences, Utah State University, Logan 84322-8700

Bovine plasmin (EC 3.4.21.7) activity on H-D-valyl-L-leucyl-L-lysyl-4-nitro-anilide was measured by determining the change in absorbance at 405 nm. Initial rates of reactions were estimated at all combinations of the following substrate concentrations [.4, 4, and 40 times the substrate concentration at one-half maximum velocity (V_max) (K_m)] and casein concentrations [.068, .68, and 6.8 times the inhibitor constant for competitive inhibition (K_I)]. By nonlinear least squares fitting of the data to an equation that described reversible enzyme kinetics, steady state kinetic parameters, maximum velocity (V_max), substrate concentration at one-half maximum velocity (V_max) (K_m), inhibitor constant for competitive inhibition (K_I), and inhibitor constant for uncompetitive inhibition (K_I) were estimated. casein fit the equation as a competitive inhibitor of bovine plasmin. This enzyme has a catalytic constant (K_cat) of .0158 change in absorbance at 405 nm/min per nM, substrate concentration at one-half maximum velocity (V_max) (K_m) of .107 mM substrate, and inhibitor constant for competitive inhibition (K_I) of .86 mg/ml of casein. Bovine plasmin activity can be measured directly in bovine milk without interference from casein.

Key Words: plasmin • inhibition • casein • enzyme kinetics

Submitted on December 10, 1990
Accepted on May 31, 1991

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