Carboxypoptidase B Activity (54)

Materials Required

1. Tris buffer; 0.2 M pH 7.7 prepared at 25° C containing 0.2 M NaCl.

2. Substrate solution; 2.0 mM. Dissolve 68.0 mg hippuryl-L-arginine in 100 ml H20. Heat to approximately 50° C with stirring to accelerate dissolution if necessary. Cool to room temperature. Prepare fresh daily. Keep in ice.

3. Enzyme solution; dissolve 0.1 mg/ml in tris buffer. Keep in ice.

Procedure


1. Adjust the spectrophotometer to 254 nm and the cell temperature to 25°C

2. Mix 1.5 ml tris-buffer and 1.5 ml substrate solution in the cell. Equilibrate to 25°C

3. Add 20 μl CPB solution, mix and determine the rate increase in absorbance, at 30 second intervals for 3 minutes. Calculate 254nm/min from the initial linear reaction.

Calculation of Specific Activity

Î, 1%, 280 = 21.2       mg/ml=A280 x 0.472      Vol = 3.02 ml       A= 254 nM        T= 25°C
Light Path = 1.0 cm
0.349 = increase in absorbency of 1 mM hippuric acid at pH 7.7

Units  =  ∆A/min x 3.02 ml
mg          0.349 x 0.002 mg