PPE assay using NS945

DETERMINATION OF PORCINE PANCREATIC ELASTASE ACTIVITY

Assay with Suc-Ala-Ala-Ala-pNA (EPC No. NS945) as substrate (44).

Materials Required

  1. 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.
     
  2. 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.
     
  3. 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.
     
  4. 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.
     

Procedure

  1. Adjust the spectrophotometer to 410 nm and cell temperature to 25ºC.
     
  2. Equilibrate 2.5 ml of  Tris buffer and 0.5 ml of substrate solution to 25º in the cell.
     
  3. 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