PROCEDURES FOR DETERMINATION OF ENZYME
ACTIVITY OF HUMAN SPUTUM ELASTASE
Assay with N-Suc-Ala-Ala-Ala-pNA as substrate (44).
- Tris-NaCl buffer; 0.1 M Tris pH 7.5 at 25º C containing 0.5 M NaCl and 0.01% NaN3. Dissolve 12.70 g Tris-HCl, 2.36g Tris-base, 29.22 g NaCl and 0.10 g NaN3 in 900 ml H2O. Determine the pH and titrate if necessary with 0.1 M HCl or 0.1 M NaOH to pH 7.5 at 25ºC. Bring to 1000 ml with water.
- Substrate solution, 1.0 mM. Dissolve 45.5 mg of N-Suc-Al-Al-Al-pNA (EPC No. NS945) in 2.0 ml 1-methyl-2-pyrrolidone. Bring to final volume of 100 ml with Tris-NaCl buffer.
- Elastase solution. Dissolve 1.0 mg per ml in (0.05 M NaOAc pH 5 containing 0.1 M NaCl). Keep cold in an ice bath.
- Adjust the spectrophotometer to 410 nm and the cell temperature to 25º C.
- Equilibrate 3.0 ml of substrate solution to 25º C in the cell.
- Add 0.01 ml of elastase solution, mix and determine the rate increase in absorbency at 2-3 minute intervals. The rate of increase should be linear for 5 to 10 minutes.
Calculation of Specific Activity
Î, 1% , 280 = 6.65 mg/ml = A280 x1.50
Vol = 3.01 ml A = 410 nm T = 25º C Light Path = 1 cm
8.8 = mM extinction coefficient of pNA at 410 nm
Units = ∆A410 x 3.01 ml
mg 8.8 x 0.01 mg