Characteristics of two trypsin type isozymes isolated from the arctic fish capelin (Mallotus villosus)

Abstract

1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.