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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 approximately 8.5 and approximately 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 pI 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.

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The proteinase inhibitors (PIs) active against bovine pancreatic trypsin, chymotrypsin, and insect midgut trypsin-like proteinases were found in the seeds of 14 cultivars and eight wild types of pigeonpea, Cajanus cajan L.. The inhibitory activity of PIs against trypsin and chymotrypsin, as well as their activity profile on gelatin-polyacrylamide gel electrophoresis (PAGE) were identical among the various cultivars. In contrast to cultivars, the wild types showed differences in inhibitory activity of PIs and their activity profile on gelatin-PAGE. The PIs from all cultivars and few wild types showed 10- to 50-fold higher activity against midgut trypsin-like proteinases of Achaea janata (L.) (Lepidoptera: Noctuidae), compared with bovine pancreatic trypsin. However, the PIs from both cultivars and wild types showed three- to nine-fold less activity against Spodoptera litura (F.) (Lepidoptera: Noctuidae) midgut trypsin-like proteinases, compared with bovine pancreatic trypsin. This inhibitory potential of PIs from cultivars and wild types, toward midgut trypsin-like proteinases from A. janata was further evident by the strong activity profile visualized on gelatin-PAGE. These results further suggest that the inhibitory potential of PIs from pigeonpea cultivars and wild types could be exploited in management of nonhost insects.

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