PURIFICATION AND COMPOSITION OF THE TRYPSIN-CHYMOTRYPSIN INHIBITORS OF PHASEOLUS VULGARIS L. VAR ROSINHA G2

Three trypsin-chymotrypsin inhibitors were purified to homogeneity from Phaseolus vulgaris L. var Rosinha G2 (Brazilian pink beans). At least one more trypsin-chymotrypsin inhibitor was present. Purfication was achieved by fractionation into albumin and globulin fractions based on differential salt/water solubilities, affinity chromatography on trypsin-Sepharose, DEAE-cellulose chromatography and CM-cellulose chromatography. Purity of the inhibitors was determined by rechromatography, combining ratios with trypsin and by disc gel electrophoresis. The isolated inhibitors were very similar, although distinguishable, by amino acid analysis. They had no tryptophan and valine, one residue of methionine, low glycine, alanine, leucine and tyrosine contents and high levels of serine, threonine, proline and half-cystine (18.5 to 21.0 residues per molecular weight of 20,000). They also contained a low amount of carbohydrate (one equivalent of mannose per 20,000 g protein).