Casein interference in bovine plasmin assays using a synthetic substrate

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