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.
Bastian
E D
ED
Department of Nutrition and Food Sciences, Utah State University, Logan 84322-8700.
Brown
R J
RJ
Ernstrom
C A
CA
D000818:Animals; D001667:Binding, Competitive; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D002863:Chromogenic Compounds; D005341:Fibrinolysin / Q000032:analysis* / Q000378:metabolism; D008892:Milk / Q000032:analysis*; D009842:Oligopeptides
D002364:Caseins; D002863:Chromogenic Compounds; D009842:Oligopeptides; C018788:valyl-leucyl-lysine 4-nitroanilide; D005341:Fibrinolysin