The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Purification and properties of the inhibitors.
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 degrees C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HCl. The intact inhibitors were destroyed when the peas were cooked at 100 degrees C or when they were toasted at 130 degrees 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).
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004355:Drug Stability; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010434:Pepsin A; D010940:Plant Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D010944:Plants / Q000032:analysis*; D013696:Temperature; D014361:Trypsin Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D014508:Urea
D000596:Amino Acids; D006146:Guanidines; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D014508:Urea; D002918:Chymotrypsin; D010434:Pepsin A