PURIFICATION AND CHARACTERIZATION OF OVINE PANCREATIC ELASTASE

Elastase was isolated from ovine pancreas and purified to homogeneity by two different procedures. One involved precipitation with ammonium sulphate, p-aminobenzamidine-Sepharose chromatography, CM-Sepharose io n exchange chromatography and S-300 Sephacryl chromatography. The othe r involved the direct adsorption of elastase by tri-L-alanyl-Sepharose chromatography and a CM-Sepharose step. The enzyme, which was produce d in an inactive form in the pancreas, was activated with a trace of t rypsin prior to chromatography. Ovine pancreatic elastase has an isoel ectric point above pI 9.3 and its molecular mass is estimated at simil ar to 25 kDa. The optimal pH range for activity is between 8.0 and 8.4 and the enzyme is unstable at pH below 4.0 and above 10.0 and at temp eratures above 65 degrees C. The kinetic properties of the enzyme were determined with succinyl-Ala-Ala-Ala-p nitroanilide as the substrate. K-m and k(cat) K-m(-1) proved to be similar to the kinetic parameters of porcine elastase determined simultaneously. (C) 1998 Elsevier Scie nce Inc. All rights reserved.