Abstract
The present work describes the purification and characterisation of a Bowman-Birk-like trypsin and chymotrypsin inhibitor from the seeds of tepary bean (Phaseolus acutifolius). Simple purification steps of ion exchange, hydrophobic interaction, and size exclusion chromatography were applied to purify a 17.8 kDa tepary bean protease inhibitor (TBPI). Analysis of the amino acid profile of the TBPI revealed that aspartic acid, glutamic acid and serine as the dominant amino acids. Stoichiometry of the reactions of trypsin and chymotrypsin with TBPI showed a molar ratio of 1:1 between the inhibitor and these enzymes. A 50% reduction in antitrypsin activity was observed when the free amino groups of TBPI were chemically modified with 2,4,6-trinitrobenzene; however, antichymotrypsin activity was not altered by this modification. These results show that purified TBPI reacts distinctly with trypsin and chymotrypsin, indicating that it might have two different reactive sites. (C) All Rights Reserved.