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

The two principal isoinhibitors P‐5 and P‐6 isolated earlier from the seeds of chick peas ( Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix‐bound trypsin are shown to be the virgin and trypsin‐modified forms of the same inhibitor. The virgin inhibitor P‐5 consists of a single peptide chain of 66 amino acid residues cross‐linked by seven disulfide bridges. Upon interaction with the matrix‐bound trypsin under the conditions used the virgin inhibitor P‐5 is about 50% converted to P‐6 by cleavage of a single Lys‐Ser linkage at position 14– 15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1–14 and 15–66 separate readily by molecular‐sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence of residues 1‐25 was established by Edman degradation. Cleavage of the Lys 1 4–Ser 1 5 linkage at the trypsin‐reactive site of the virgin inhibitor significantly reduces its trypsin inhibitory activity without affecting its anti‐chymotryptic activity. Upon treatment with carboxypeptidase B the trypsin‐modified inhibitor loses its anti‐tryptic activity but remains fully active against chymotrypsin. A similar treatment has no effect on the virgin inhibitor. Furthermore, the complex of the inhibitor with trypsin is inactive towards trypsin but remains fully active against chymotrypsin and vice versa. It is thus concluded that the principal inhibitor in chick peas inhibits both enzymes simultaneously and is clearly of the ‘double‐headed’ type with separate and independent reactive sites for the two enzymes. The molecule appears to be highly homologous to the Bowman‐Birk soybean inhibitor and to the lima bean inhibitor.