Fractionation and Properties of Trypsin and Chymotrypsin Inhibitors from Lima Beans

Abstract

Abstract Inhibitors of trypsin and chymotrypsin from several highly inbred genetic types of lima beans were fractionated by ion exchange chromatography. Six chromatographically distinct inhibitors were found in all cases. However, the relative distribution of these components fell into two types, dependent upon the variety of bean used. One milligram of one of the chromatographically homogeneous fractions inhibited 2.6 mg of bovine trypsin and 1.2 mg of α-chymotrypsin. The presence of a double-headed inhibitor was indicated, in which both inhibitory sites were present on the same molecule. The molecular weights of this fraction calculated from the amino acid composition and from the stoichiometry of trypsin inhibition were 9,670 and 9,050 g per mole, respectively. However, a molecular weight of 16,200 g per mole was found by the Ehrenberg method, and an s20,w value of 1.81 S was obtained. This indicates a possible aggregation or a molecular weight twice that indicated by the minimum one from amino acid analyses. Chemical modification of the amino groups of this fraction with trinitrobenzenesulfonic acid completely destroyed the trypsin-inhibitory activity, with no effect on the chymotrypsin-inhibitory activity. Amidination of the amino groups, which results in no change in charge, also greatly lowered the trypsin-inhibitory activity. The results of the chemical modifications indicate that a fast reacting lysine residue is critical for the trypsin-inhibitory activity.