HSE assay using NS945

PROCEDURES FOR DETERMINATION OF ENZYME

ACTIVITY OF HUMAN SPUTUM ELASTASE 

Assay with N-Suc-Ala-Ala-Ala-pNA as substrate (44).

Materials Required

  1. 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.
     
  1. 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.
     
  1. 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.


Procedure

  1. Adjust the spectrophotometer to 410 nm and the cell temperature to 25º C.
     
  1. Equilibrate 3.0 ml of substrate solution to 25º C in the cell.
     
  1. 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