Proteinase inhibitor from Vicia angustifolia L. var. segetalis koch. Isolation, characterization, and chemical modification.:Isolation, Characterization, and Chemical Modification

A proteinase inhibitor which inhibits trypsin [EC 3. 4. 21. 4] and α-chymotrypsin [EC 3. 4. 21. 1] was isolated from the seeds of Vicia angustifolia L. var. segetalis Koch and purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-Sephadex A-25. The inhibitor has a molecular weight of about 13, 000 when determined by molecularsieve chromatography on Sephadex G-100 and 12, 600 from the amino acid composition, which revealed high contents of half-cystine, aspartic acid, serine, and glutamic acid residues. The absence of free sulfhydryl groups, tryptophan, and carbohydrate was also observed. The amino- and carboxyl-terminal residues of the inhibitor were determined to be glycine and asparagine, respectively, suggesting that this inhibitor is composed of a single polypeptide chain. The inhibitor has an isoelectric point at pH 7.6 and is considerably stable in acidic and neutral pH regions, however, it loses inhibitory activities at alkaline pH.<br> Stoichiometry of the reactions between the inhibitor and the enzymes showed that this inhibitor reacted with trypsin in a 1:1 and with chymotrypsin in a 1:2 molar ratio.<br> Maleylation of the inhibitor revealed that even when all the 9 amino groups, i.e. one α-amino and 8 ε-amino groups, were modified, the inhibitor retained its full antitryptic activity. On the other hand, on 1, 2-cyclohexanedione treatment, the inhibitor lost almost all the antitryptic activity and also 50% of the antichymotryptic activity. Modifications of all the three tyrosyl residues in the inhibitor decreased the antichymotryptic activity by 50% but had no effect on the antitryptic activity. The results of these chemical modifications seem to support that this inhibitor has one reactive site for trypsin and two for α-chymotrypsin.