Isolation and characterisation of four trypsin-chymotrypsin inhibitors from lentil seeds

Abstract

Twenty‐three proteinase inhibitors were isolated from Syrian local small lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin‐Sepharose. They all inhibited human and bovine trypsin and chymotrypsin. Three inhibitors (LCI‐11·7, ‐3·3 and ‐4·6) were separated and purified to homogeneity by anion exchange chromatography and preparative isoelectric focusing (IEF) with immobilised pH gradients; a fourth (LCI‐2·2) required additional reversed‐phase high‐pressure liquid chromatography. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of free sulphydryl groups, methionine and tryptophan. The calculated minimum number of amino acid residues per molecule, the calculated molecular masses confirmed by gel liquid chromatography, gel‐permeation high‐pressure liquid chromatography and sodium‐dodecylsulphate polyacrylamide gel electrophoresis, and the isoelectric points determined by IEF (immobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5·26, 5·88, 6·80 and 7·80 for LCI‐1·7, ‐2·2, ‐3·3 and ‐4·6, respectively. All four inhibitors inhibited human trypsin less than bovine trypsin, and human chymotrypsin more than the bovine enzyme. All these properties are in accordance with the classification of the four lentil inhibitors as members of the Bowman‐Birk proteinase inhibitor family. © 1998 Society of Chemical Industry.

In vitro protein digestibility and physico-chemical properties of flours and protein concentrates from two varieties of lentil (Lens culinaris)

“…The Bowman-Birk inhibitors are composed of a single chain having a MM of 8 to 10 kDa with a high cysteine content (14 residues); these inhibitors form intra-chain disulphide bridges and have reactive sites, one for trypsin and one for chymotrypsin which can reduce the activity of these enzymes when bound. 68,69 Lentil seeds contain mainly Bowman-Birk trypsin inhibitors. 68 A trypsin/chymotrypsin Bowman-Birk inhibitor consisting of 67 amino acid residues and having a MM of 7.448 kDa was isolated from lentil seeds.…”

“…68,69 Lentil seeds contain mainly Bowman-Birk trypsin inhibitors. 68 A trypsin/chymotrypsin Bowman-Birk inhibitor consisting of 67 amino acid residues and having a MM of 7.448 kDa was isolated from lentil seeds. The isolated trypsin inhibitor shows dissociation constants of 0.54 and 7.25 nM for trypsin and chymotrypsin, respectively.…”

Unveiling the contribution of Osborne protein fractions to the physicochemical and functional properties of alkaline and enzymatically extracted green lentil proteins

“…This suggests that perhaps due to the nature of the sequential process, which employed milder conditions in the initial stages (25°C, approximately neutral pH), the Kunitz‐type protein inhibitor was not as efficiently extracted, causing it to be distributed among all fractions in small amounts that were too low to detect. Notably, Kunitz‐type proteins in lentils are inherently low in concentration as lentil protease inhibitors are primarily the Bowman‐Birk type (Belitz & Weder, 1990; Weder & Kahleyß, 1998). However, there were a few confounding factors that made the definitive identification of this protein difficult.…”