ISOLATION AND CHARACTERIZATION OF 4 TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM LENTIL SEEDS

Twenty-three proteinase inhibitors were isolated from Syrian local sma ll lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin-S epharose. They all inhibited human and bovine trypsin and chymotrypsin . Three inhibitors (LCI-1.7, -3.3 and -4.6) were separated and purifie d to homogeneity by anion exchange chromatography and preparative isoe lectric focusing (IEF) with immobilised pH gradients; a fourth (LCI-2. 2) required additional reversed-phase high-pressure liquid chromatogra phy. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of f ree sulphydryl groups, methionine and tryptophan. The calculated minim um number of amino acid residues per molecule, the calculated molecula r masses confirmed by gel liquid chromatography, gel-permeation high-p ressure liquid chromatography and sodium-dodecylsulphate polyacrylamid e gel electrophoresis, and the isoelectric points determined by IEF (i mmobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5.26, 5.88, 6.8 0 and 7.80 for LCI-1.7, -2.2, -3.3 and -4.6, respectively. All four in hibitors inhibited human trypsin less than bovine trypsin, and human c hymotrypsin more than the bovine enzyme. All these properties are in a ccordance with the classification of the four lentil inhibitors as mem bers of the Bowman-Birk proteinase inhibitor family. (C) 1998 Society of Chemical Industry.