Clostripain (Endoproteinase-Arg-C) - Assay

Adjust spectrophotometer to 253 nm and 25°C.

Pipette into each cuvette as follows:

0.075 M phosphate buffer, pH 7.6	1.0 ml
7.5 mM DTT	1.0 ml
0.75 mM BAEE	1.0 ml

Incubate in spectrophotometer for 3-5 minutes to achieve temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme and record A₂₅₃ for 4-5 minutes. Determine ΔA₂₅₃/minute from the linear portion of the curve. Note: The reaction appears to be most linear with respect to enzyme concentration when ΔA₂₅₃/min is between 0.007 and 0.030.