DETERMINATION OF PORCINE PANCREATIC ELASTASE ACTIVITY
Assay with Suc-Ala-Ala-Ala-pNA (EPC No. NS945) as substrate (44).
- Tris buffer; 0.1 M Tris pH 8.3 at 25ºC. Dissolve 6.75 g Tris-HCl and 8.14 G Tris base in 900 ml H2O. Determine pH at 25ºC. Titrate if necessary to pH 8.3 with 0.1 M HCl or 0.1 M NaOH. Dilute to 1000 ml with H2O.
- NaOAc-NaCl buffer; 0.05 M NaOAc pH 5, containing 0.1 M NaCl. Combine 14.8 ml of 0.2 M HAc and 35.2 ml of 0.2 M NaOAc and 100 ml 0.2 M NaCl. Bring to 200 ml with H2O. Titrate to pH 5 at 25ºC.
- Substrate solution; 2.5 mM in 0.1 M Tris pH 8.3. Utilizing a 25 mg vial of N-Suc-Ala-Ala-Ala-pNA (EPC No NS945), dissolve the contents with 22 ml of tris buffer. (Note: Use about 10 ml of the buffer for 5 flushes of the substrate vial.) Dissolve with stirring. Store at 5ºC.
- Elastase solution; dissolve 1.0 mg per ml in the NaOAc-NaCl buffer. Prepare a secondary solution of 0.10 mg per ml in the same buffer. Keep both solutions cold in an ice bath.
- Adjust the spectrophotometer to 410 nm and cell temperature to 25ºC.
- Equilibrate 2.5 ml of Tris buffer and 0.5 ml of substrate solution to 25º in the cell.
- Add 0.005 ml of the 0.10 mg ml elastase solution, mix and determine the rate increase in absorbency at 1 minute intervals. The rate increase should be ca. 0.025 – 0.040 ∆410 nm per minute.
Calculation of Specific Activity
Î, 1%, 280 = 19.5 for porcine pancreas elastase
mg/ml = A280 x 0.51
Vol = 3.005 ml A=410 nm T=25ºC Light Path=1.0 cm
8.8=mM extinction coefficient of pNA at 410 nm
Units = ∆A410 x 3.005 ml
mg 8.8 x 0.0005 mg