TRYPSIN AND CHYMOTRYPSIN INHIBITOR FROM CHICK PEAS Selective Chemical Modifications of the Inhibitor and Isolation of Two Isoinhibitors

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Abstract
The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 m – 0.01 m and in KOH 0.01 m – 0.05 m even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleylation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.