Detection and partial characterisation of subtilisin inhibitors in legume seeds by isoelectric focusing

Seed extracts of ten legume species were subjected to isoelectric focusing in acrylamide gels and inhibitors of subtilisin, three other bacterial proteases, trypsin and chymotrypsin were detected by a negative-staining technique based on the chromogenic substrate, acetyl-D, L-phenylalanine-2-naphthylester. In addition to complex patterns of trypsin and chymotrypsin inhibitor zones, most legume seeds examined exhibited one major and one minor inhibitor of subtilisin, and the other bacterial proteases. However, in cowpea and black gram only the major, and in lentil and soya bean only two minor subtilisin inhibitor zones were detected. Isoelectric points of the subtilisin inhibitors range from 4.4 to 5.9. The inhibitor patterns obtained by isoelectric focusing of extracts mixed with bacterial protease confirmed that inhibitors of the bacterial proteases represent a new type of inhibitor different from the well characterised trypsin and chymotrypsin inhibitors.