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

Weder, J.K.P., Kahelyss, R.
Journal of the science of food and agriculture 1998 v.78 no.3 pp. 429-434
ion exchange chromatography, trypsin, trypsin inhibitors, humans, enzyme activity, chymotrypsin, cattle, isoelectric point, chemical structure, high performance liquid chromatography, amino acid composition, molecular weight, lentils, isoelectric focusing
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-1.7, -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.