Clostripain (Endoproteinase-Arg-C) Assay

Method: The reaction velocity is measured as an increase in absorbance at 253 nm resulting from the hydrolysis of N-benzoyl-L-arginine ethyl ester. One unit hydrolyzes one micromole of BAEE per minute at 25°C and pH 7.6 under the conditions specified.

Reagents

• 0.075 M Sodium phosphate buffer, pH 7.6
• 7.5 mM Dithiothreitol (DTT)
• 0.75 mM N-Benzoyl-L-arginine ethyl ester (BAEE)
• 1.0 mM Calcium acetate containing 2.5 mM dithiothreitol (activation solution)

Enzyme

Dissolve or dilute the enzyme at a concentration of 1 mg/ml in water. Immediately prior to assay, dilute the enzyme further in 1.0 mM Calcium acetate containing 2.5 mM dithiothreitol to a concentration of 0.2-0.8 units/ml.

Procedure

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.

Calculation