Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae.

1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.