Bowman-Birk Proteinase Inhibitor from Cajanus cajan Seeds: Purification, Characterization, and Insecticidal Properties

Abstract

A red gram proteinase inhibitor (RgPI) was purified from red gram (Cajanus cajan) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of similar to 8.5 and similar to 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with p/ values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence `DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family, RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta. Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.