Trypsin - Assay
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.
- 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
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:
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.
where 540 is the extinction coefficient of p-toluenesulfonyl-L-arginine at 247 nm
1 TAME Unit = 19.2 USP/NF Units = 57.5 BAEE Units.