DETERMINATION OF HUMAN SPUTUM ELASTASE ACTIVITY (62)
Assay with MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) as substrate.
1. Tris-NaCl buffer; 0.1 M Tris pH 7.5 at 25º C containing 0.5 M NaCl. Dissolve 12.70 g Tris-HCl, 2.36g Tris-base and 29.22 g NaCl 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 H2O.
2. Substrate solution 0.2 mM. Dissolve 12.0 mg Meo-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) in 1.0 ml of 1-methyl-2-pyrrolidone. Bring to a final volume of 100 ml with Tris-NaCl buffer.
3. Elastase solution. Dissolve 1.0 mg per ml in 0.05 M NaOAc pH 5 containing 0.1 M NaCl. Prepare a secondary dilution of 0.10 mg per ml in the same buffer. Keep both solutions cold in an ice bath.
1. Adjust the spectrophotometer to 410 nm and cell temperature to 25º C.
2. Equilibrate 3.0 ml of substrate solution to 25º C in the cell.
3. Add 0.01 ml of elastase solution (0.10 mg/ml), 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
Units = ∆A/min x 3.01 ml
mg 8.8 x 0.001 mg