Purification and Some Physical Properties of a Chymotrypsin‐Like Protease of the Larva of the Hornet, Vespa orientalis

Abstract

A chymotrypsin-like endopeptidase has been purified by ion-exchange chromatography, affinity chromatography, and gel filtration. The enzyme preparation is homogeneous in the ultracentrifuge and disc electrophoresis. The enzyme is proved to be free from any other proteolytic activities. The molecular weight of the proteinase as determined with several techniques (ultracentrifugation, gel filtration and electrophoresis without dodecylsulfate) is in the range between 13 000-14000 ; however, by dodecylsulfate gel electrophoresis a molecular weight of 23 000 was obtained. The obtained physical constants of hornet chymotrypsin are : sedimentation coefficient s ; , ,~ = 1 . 9 6~ s, diffusion coefficient D:o,w = 131 pm2 s-l, partial specific volume B = 0.737 ml g-l, frictional ratio f/fmin = 1.04, and degree of hydration = 0.1 g per g protein.During our investigation of the evolution of endopeptidases from invertebrates, Sonneborn et al. [I] have characterized a prot.einase from the midgut of the larva of the hornet, Vespa orientalis. This protease is homologous with mammalian chymotrypsin by such criteria as the complete inhibition by phenylmethylsulphonyl fluoride and Nu-tosyl-Lphenylalanine chloromethane and the cleavage specificity. However, by gel filtration on Sephadex the molecular weight was found to be 12 800. This molecular weight is much lower than that of previously known chymotrypsins from vertebrates and other invertebrates. Therefore the question arises, whether it is a true molecular weight or an artefact which could be result from interactions between Sephadex gel and the protein.The present paper describes a modified purification method of the hornet chymotrypsin and reports a study of its size and shape.

Insect Proteases and Peptidases

“…A combination of chromatography on cation-cxchange columns and affinity chromatography yielded a pure enzymc of molecular weight 13,000-14,000-a much smaller molecule than most known chymotrypsins (29). T h e molecular weight was detcrmined by several methods-ultracentrifugation, gel filtration, polyacrylamide gel electrophoresis with and without SDS (29), and stoichiometric reactions with isotopically labeled PMSF and ZPCK (30)-all with very good agreement. Amino acid analysis showed that the enzyme contained only four cysteine residues and no methionine (30).…”

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants

“…The molecular masses found are those usual for chymotrypsins (Terra and Ferreira, 1994) and the pH optima determined for the different chymotrypsins conform to what is expected from the taxa they belong to (Terra and Ferreira, 1994). The same purification procedure described above has been used in several instances (Jany et al, 1974;Sakal et al, 1988;Peterson et al, 1995;Botos et al, 2000;Muharsini et al, 2001), although there are reports regarding non-lepidopteran insects in which a successful purification of chymotrypsins was attained by ion-exchange chromatography (Terra and Ferreira, 2005). It is worth-mentioning that Elpidina et al (2005) purified a chymotrypsin from T. molitor midgut that differs from the one described here in being stable during ion-exchange chromatography.…”

Enzymic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro

“…The protease VCP II was purified from the larval midguts as described previously (Jany et al, 1974a), except that CM-Sephadex C-25 was replaced by CM-Sepharose C1-6B and Sephadex G-75 by Sephadex G-50. Protein Determination.…”