Lima Bean Protease Inhibitor: Reduction and Reoxidation of the Disulfide Bonds and Their Reactivity in the Trypsin–inhibitor Complex

Abstract

Lima bean protease inhibitor is a protein which inhibits both trypsin and chymotrypsin at different and independent sites. Complete reduction of the disulfide bonds of the inhibitor results in loss of biological activity. By air oxidation of the reduced inhibitor, full chymotrypsin inhibitory activity and up to 50% of the trypsin inhibitory activity can be regained; the rates at which these activities are regained were different. Attempts to obtain selective cleavage of one or a few disulfide bonds by carefully controlling reaction conditions were unsuccessful. All the disulfide bonds of lima bean inhibitor appear equally accessible to the reagent and with a less than twofold molar excess of dithioerythritol over the molarity of disulfide bonds, complete reduction was obtained; both inhibitory activities were equally sensitive to reduction and were lost as a linear function of the average number of disulfide bonds reduced and carboxymethylated. In contrast to this, the disulfide bonds of lima bean inhibitor are stabilized when the inhibitor is in the form of a molar complex with trypsin. Under these conditions, only one out of a possible eight disulfide bonds could be reduced in the inhibitor with up to a 10-fold molar excess of dithioerythritol. The modified inhibitor obtained after dissociation of reduced and carboxymethylated trypsin–inhibitor complex appeared fully active against both trypsin and chymotrypsin.