The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)

Abstract

The six isoinhibitors of trypsin and chymotrypsin which were previously purified from a crude extract of chick peas were further studied with the intention of establishing the relationships among them. Exposure of each fraction to active, matrix-bound trypsin resulted in a decrease of the specific inhibitory activities towards trypsin of fractions P-1, P-3 and P-5 while those of P-4 and P-6 were increased. Each of these fractions was separated into several sub-fractions after chromatography on a DEAE-Sephadex A-25 column under conditions identical to those used previously for their purification. The elution positions, specific inhibitory activities towards trypsin and electrophoretic patterns in polyacrylamide gels of the various sub-fractions were determined. On the basis of the results thus obtained it is concluded that fractions P-3, P-4, P-5 and P-6 originate from a common inhibitor molecule. From the amino acid compositions and aminoterminal sequences of these four fractions, it is further concluded that P-3 and P-4 are identical proteins and that P-5 and P-6 are probably derived from P-3 and P-4, respectively, as a result of tryptic removal of the amino-terminal Val-Lys residues from the latter two fractions. Since the distribution of the products obtained from P-1 is different from that obtained from the other four fractions, it is also concluded that P-1 is propably derived from a second inhibitor originally present in chick peas.The inhibitors in chick peas were also isolated by conventional chromatographic procedures. Preliminary results indicate the presence of two inhibitors, both with a molecular weight of around 16000. Since the fractions thus obtained were not homogeneous they were not further characterized. Nevertheless, the findings confirm the conclusions reached above and also show that the inhibitors probably exist as dimers in the seeds of chick peas since each of the isoinhibitors previously isolated had molecular weights of about 10000.A large majority of plant and animal tissues from which protein proteinase inhibitors have been isolated contain more than one inhibitor having similar or different structures and/or functions [l, 21. The soybean contains at least four different inhibitors [3] while the lima bean contains six isoinhibitors [4] which are very similar to each other. The presence of multiple proteinase inhibitors in the same plant or animal tissue has been ascribed to genetic heterogeneity [5,6] or to differences in amide content [7,8]. In those cases where the inhibitors have been isolated by selective binding to matrix-bound trypsin or chymotrypsin [9-111 the multiplicity of inhibitors is in- creased by the presence of the modified form of each inhibitor in the material that is dissociated from the coupled enzyme. In a previous report [12] the purification and properties of six isoinhibitors from chick peas was described. On the basis of their different inhibitory activities towards bovine trypsin and the fact that they were isolated by affinity chromatography on trypsinSeph...

Proteinase Inhibitors from Pea Seeds: Purification and Characterization

“…During purification procedures, partial proteolysis of the reactive site and modification by proteolytic cleavage of further peptide linkages may occur if affinity chromatography is performed on trypsin-bound gels (Belew, 1977;Kortt, 1979;Gatehouse et al, 1980;Mahoney et al, 1984). Even if an inactivated protease such as anhydrotrypsin is grafted to the affinity column, the pH shock generally used for eluting the bound inhibitors may modify their structure; especially, deamidation may be induced by extreme pH, leading to incorrect molecular mass values and sequences.…”