The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)

Abstract

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50 % of the isolated inhibitors and were further purified to a homogeneous state.The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 "C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HC1. The intact inhibitors were destroyed when the peas were cooked at 100 "C or when they were toasted at 130 "C.The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20 % of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).Proteins which inhibit the activity of proteolytic enzymes have been isolated from the tissues of a variety of animal and plant species. Their distribution, properties and biological significance are presented in recent general reviews [ 1,2] and proceedings of research conferences [3,4]. While proteinase inhibitors in most leguminous seeds have been extensively studied, those from Cicer arietinum (commonly called chick peas, bengal gram, garbanzo or coffee pea) have so far not been purified and characterized, although Borchers and Ackerson [5] reported in 1947 that these seeds contain a high concentration of trypsin inhibitor. Their results were confirmed by Sohonie and Bhandarkar [6] who also showed that the crude inhibitor preparation was acid stable and fairly thermostable. Later, Abramova and Chernikov [7] showed that extracts from chick peas inhibited trypsin and chymotrypsin but not pepsin.Since chick peas constitute an important source of dietary protein in some parts of the world, and in view of the limited investigation carried out on their inhibitors, we have undertaken to purify and study them in detail. Such studies can contribute, at least in part, to a understanding of their significance in animal nutrition. In this paper the purification of the inhibitors including some of their physical and chemical properties is described. The...

The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)

“…The inhibitors in chick peas were isolated from an aqueous extract of mature seeds by affinity chromatography on trypsin-Sepharose 6B and further separated into six active fractions by ion-exchange chromatography as described previously [12]. The chromatographic fractions are referred to as P-1 to P-6 in order of their elution from the column.…”

“…The concentration of the inhibitors in solution was determined from their absorbances at 275 nm using A: ?& of 5.40 [12]. In the case of trypsin, an A: c% at 280 nm of 15.6 was used.…”

“…The coupling process and all subsequent operations were done at 4 "C as outlined elsewhere [12,21]. The trypsin-Sepharose 6B column (5 x 10 cm) had a capacity to adsorb about 100 mg of chick pea inhibitors.…”

“…In those cases where the inhibitors have been isolated by selective binding to matrix-bound trypsin or chymotrypsin [9-111 the multiplicity of inhibitors is in- creased by the presence of the modified form of each inhibitor in the material that is dissociated from the coupled enzyme. In a previous report [12] the purification and properties of six isoinhibitors from chick peas was described. On the basis of their different inhibitory activities towards bovine trypsin and the fact that they were isolated by affinity chromatography on trypsinSepharose 6B, we postulated that three of the inhibitors represented the modified forms of the remaining three fractions.…”

“…In a previous report [12] the purification and properties of six isoinhibitors from chick peas was described. On the basis of their different inhibitory activities towards bovine trypsin and the fact that they were isolated by affinity chromatography on trypsinSepharose 6B, we postulated that three of the inhibitors represented the modified forms of the remaining three fractions.…”