Purification and characterization of a chymotrypsin-like enzyme (protease-S) in thermally injured rat skin.

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 30 000. 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 micrometer), 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 proteases-S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.