Photoreactive, active derivatives of trypsin and chymotrypsin inhibitors from soybeans and chickpeas.

Abstract

The photoreactive arylsufenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (STI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-STI indicated that only one of the two tryptophans, 93 or 117, present in STI was modified. Amino acid analysis of the two separated CNBr-cleavage products of 2,4-NAPS-STI showed that only tryptophan 93 underwent modification. 2,4-NAPS-STI fully retained its inhibitory activity against trypsin. The covalent attachment of 2,4-NAPS-STI to tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride. Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas were prepared by selective modification of the epsilon-amino groups of 2,4(5)-NAPS-Cl. The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.