INHIBITORS OF HUMAN AND BOVINE TRYPSIN AND CHYMOTRYPSIN IN FENUGREEK (TRIGONELLA-FOENUM-GRAECUM L) SEEDS - DEMONSTRATION AND PURIFICATION

Fenugreek contained proteinase inhibitors inhibiting 5-9 mg human trypsin, 5-7 mg bovine trypsin, 2-6 mg human chymotrypsin, and 1-3 mg bovine chymotrypsin per g seed material. About 30 inhibitors were electrophoretically detected, and 23 of them, inhibiting all the four enzymes, were characterized by means of their isoelectric points: a group of acid inhibitors (TFI-A1 to A10, pI4.48-5.12), a group of neutral inhibitors (TFI-N1 to -N6, pI 5.91-6.71), and a group of basic inhibitors (TFI-B1 to -B7, pI 7.76-9.77). To eliminate the galactomannans which complicate further purification, coarsely ground seeds were separated by density into two fractions, seed coats + endosperm and cotyledons + embryos (C + E). Isolation of the fenugreek inhibitors by extraction of fraction C + E, followed by ammonium sulfate fractionation and affinity chromatography on anhydrotrypsin-Sepharose, resulted in an about 700-fold enrichment.