PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

• Published in: Journal of food biochemistry Vol. 5; no. 3; pp. 215 - 232
• Main Authors: SGARBIERI, VALDEMIRO C., WHITAKER, JOHN R.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.09.1981
• ISSN: 0145-8884; 1745-4514
• DOI: 10.1111/j.1745-4514.1981.tb00674.x

ABSTRACT The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G‐100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10−7 cm2s−1, a partial specific volume of 0.69 cm3g−1 and a molar absorptivity of 5.5 × 103 M−1 cm−1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The Ki values for trypsin were: A, 8.5 × 10−10 M; B, 1.8 × 10−10 M and C, 6.8 × 10−10 M while for chymotrypsin they were: A, 4.4 × 10−7 M; B, 2.8 × 10−8 M and C, 3.0 × 10−8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide‐treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.