DETERMINATION OF HUMAN SKIN CHYMASE ACTIVITY
Assay with N-Benzoyl-L-Tyrosine-Ethyl Ester as substrate (86)
Materials required
1. Buffer; 0.6 M Tris-HCl pH 8.0 containing 3.0 M NaCl at 25°C. Dissolve 53.3 gm Tris-HCl, 31.8 gm Tris-base and 175.3 gm NaCl with 900 ml H2O. Titrate to pH 8.0 at 25°C, bring to 1000 ml with H2O.
2. Substrate; 1.0 mM. Dissolve 31 mg N-Benzoyl-L-Tyrosine-Ethyl Ester in 100 ml of 30% (v/v) ethanol in H2O.
3. Enzyme solution. Prepare 0.1 mg per ml in 0.05 M NaOAc H 5.0. Keep at 5°C in ice bath.
Procedure
1. Adjust the spectrophotometer to 256 nm and the cell temperature to 25°C.
2. Pipette 1.5 ml of buffer and 1.5 ml of substrate into the cell. Mix, equilibrate to 25°C in the cell.
3. Add 10 μl enzyme solution; mix and record increase in A256 nm/min for 2-4 minutes.
Calculation of Specific Activity
Vol = 3.01 ml A= 256 nm T= 25°C Light Path = 1 cm Î, 1 mM Tyrosine = 0.964
Units = ∆A256/min x (3.01 ml)
mg 0.964 x 0.001 mg