Method: Various assays have been described in the literature. Spencer et al. (1975) report an assay using as substrate an intact protein to which has been attached a fluorescent dye, 1-anilino-8-naphthalenesulfonate (ANS). Stewart (1973) has described a method using chromogenic substrates N-benzoyl-DL-arginine p-nitroanilide (DL-BAPA), N-glutaryl-L-phenylaline p-nitroanilide (L-GPNA). Ford et al. (1973) report on the use of P-nitro-phenyl p-guanidino benzoate (NPGB) to determine active site exposure of immobilized trypsin, a stable acylated enzyme is formed, plus p-nitrophenol spectrophotometrically determined at 410 nm. The assay used in the Worthington laboratory is described below.
One unit hydrolyzes 1 umole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at 25°C, pH 8.2, in the presence of 0.01 M calcium ion.
Dilute to a concentration of 10-20 ug/ml in 0.001 N HCl.
Set spectrophotometer at 247 nm and 25°C.
Pipette into each cuvette as follows:
Incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.1 ml diluted enzyme and record A247 for 3-4 minutes. Determine ΔA247 from initial linear portion of the curve. The reaction remains linear to an A247 of about 0.320. The reaction should be linear for at least three minutes. If this is not so repeat using less enzyme.
1 TAME Unit = 19.2 USP/NF Units = 57.5 BAEE Units.