Trypsin Assay

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.

Reagents

• 0.046 M Tris⋅HCl buffer, pH 8.1 with 0.0115 M calcium chloride
• 0.01 M TAME (p-toluene-sulfonyl-L-arginine methyl ester)
• 0.001 N HCl

Enzyme

Dilute to a concentration of 10-20 ug/ml in 0.001 N HCl.

Procedure

Set spectrophotometer at 247 nm and 25°C.

Pipette into each cuvette as follows:

0.046 M Tris⋅HCl buffer, pH 8.1	2.6 ml
0.01 M TAME	0.3 ml

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.

Calculation

1 TAME Unit = 19.2 USP/NF Units = 57.5 BAEE Units.