Purification and Characterization of a Chymotrypsin‐Like Enzyme (Protease‐S) in Thermally Injured Rat Skin

Abstract

Protease‐S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G‐50, CM‐Sephadex (A‐50), Sephadex G‐75 gel filtration. The optimum pH of this enzyme was 8.6–8.8, and the molecular weight determined by Sephadex G‐75 gel filtration was approximately 30000. This enzyme is active on the N‐acetyl‐L‐tyrosine ethyl ester, N‐succinyl‐L‐phenylalanine‐p‐nitroanilide (of chymotrypsin substrate) but not N‐tosyl‐L‐arginine methyl ester, N‐benzoyl‐L‐arginine‐p‐nitroanilide. Also, protease‐S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 μM), and N‐tosyl‐L‐phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin‐like proteases in mast cells and leukocyte granules. When protease‐S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation. Copyright © 1979, Wiley Blackwell. All rights reserved