alpha-chymotrypsin and trifluoromethanesulfonic-acid

Turkey ovomucoid is an inhibitor of both trypsin and chymotrypsin. Treatment of this glycoprotein with trifluoromethanesulfonic acid in anisole resulted in time-dependent removal of carbohydrate and altered its biological activity. After 6 h of treatment the apparent molecular mass obtained by SDS-PAGE decreased from 38 to 30 kDa. Carbohydrate analyses indicated loss of 94% of original saccharide residues. The inhibitory activity of each domain was analyzed independently by comparing enzymic activity of trypsin and chymotrypsin in the absence of inhibitor to that preincubated in the presence of varying amounts of native or deglycosylated ovomucoid, respectively. The results demonstrated that removal of saccharides with trifluoromethanesulfonic acid differentially affects the inhibitor activities of turkey ovomucoid. Decreased inhibitory activity of the trypsin domain was observed with casein and benzoyl arginine ethyl ester as substrates. In contrast, enhanced inhibitory activity of the chymotrypsin domain was observed with benzoyl tyrosine ethyl ester and methyl-O-succinyl-Arg-Pro-Tyr-p-nitroanilide, good substrates for chymotrypsin, but not with casein.

Topics: Animals; Carbohydrates; Caseins; Chymotrypsin; Glycosylation; Mesylates; Models, Chemical; Ovomucin; Substrate Specificity; Trypsin Inhibitors; Turkeys

Monoclonal antibodies (MAbs) are useful reagents for the study of the structure and evolution of specific epitopes. Two MAbs of IgG1 isotype, Tf-1 and Tf-2, which bind human transferrin have been produced and characterized. Both specifically recognize transferrin on immunoblots of serum. Proteolytic digestion with papain or chymotrypsin destroys the epitope recognized by Tf-1 but not Tf-2, demonstrating that the MAbs recognize distinct epitopes. Both epitopes are not recognized after treatment with 2-mercaptoethanol, suggesting that disulfide bond dependent tertiary structure is necessary for epitope integrity. Removal of carbohydrate moieties by treatment with trifluoromethane sulfonic acid likewise results in loss of reactivity. Neither MAb reacts with transferrin of mouse, rabbit or bovine origin. Both were tested for reactivity to a total of ten primate transferrins and showed different patterns of reaction. Tf-2 recognized human, chimpanzee and gorilla transferrins, whereas Tf-1 reacted with all Old World monkeys and one of three New World monkeys tested. Thus, Tf-1 and Tf-2 recognize transferrin epitopes with differential phylogenetic conservation and which are dependent not only on primary aminoacid sequence, but also upon tertiary structure and glycosylation.

Topics: Antibodies, Monoclonal; Antibody Specificity; Chymotrypsin; Epitopes; Humans; Mesylates; Oxidation-Reduction; Papain; Phylogeny; Species Specificity; Transferrin

The borate-insoluble chitin-protein complex, CB-I, from prepupal sarcophagid larvae was cleaved with chymotrypsin and trifluoromethanesulfonic acid releasing a polypeptide fragment of Mr 68 000. The intact glycoprotein was blocked at the C terminus; the N-terminal sequence of Asp-Val-Ala-His-Tyr was not homologous with seven of the borate-soluble nonglycosylated structural proteins. Bityrosine was identified as a component of the primary chain, both half-residues occupied in peptide linkages. Sclerotization initiated a decline in bityrosine coincident with the addition of soluble proteins to the tanned matrix. The chitin-protein complex also included bound peroxidase, propolyphenol oxidase, and an o-diphenol subject to oxidation on activation of the zymogen. In the course of the oxidation N termini declined in accordance with the formation of 1,4 quinonoid cross-links.

Topics: Amino Acid Sequence; Chitin; Chymotrypsin; Diptera; Glycoproteins; Larva; Mesylates; Molecular Weight; Proteins; Solubility