Abstract
A crude preparation of proteinase inhibitors of navy beans was found by ion-exchange chromatography to contain at least two inhibitors. Particular attention was given to the purification of one of these by chromatographic and electrophoretic methods. Details of selected procedures for the isolation of the inhibitor identified as chromatographic component V are described.
Preincubation of this inhibitor with trypsin at pH 3 prior to addition of a substrate at a pH favorable for substrate hydrolysis blocks the esterolytic but not the proteolytic activity of this enzyme. The differential effect of low pH preincubation is greater than with other inhibitors used for comparison.
In contrast with the low pH preincubation effects obtained with the navy bean inhibitor and trypsin, no such differential effect on the proteolytic and esterolytic activities of α-chymotrypsin were observed. An altered electrophoretic pattern and nonhomogeneity of the navy bean inhibitor after low pH preincubation with trypsin is consistent with the hypothesis that alteration of the inhibitor accounts, in part, for its subsequent differential effect on the esterolytic and proteolytic activities of this enzyme.
The constant apparent specific activity of the navy bean inhibitor with different lots of trypsin containing varying proportions of active and inactive enzyme suggests that this inhibitor, unlike others, reacts with inactive as well as active trypsin.
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This work was supported in part by grants from the United States Public Health Service (SO1-FR-05371-8) and the Indiana Heart Association.