Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea.

Abstract

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216\u202fDa) and RsKI (19,412\u202fDa) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki\u202f=\u202f128.5\u202f±\u202f4.5\u202fnM) and chymotrypsin (Ki\u202f=\u202f807.8\u202f±\u202f23.7\u202fnM) while RsKI (Ki\u202f=\u202f172.0\u202f±\u202f9.2\u202fnM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100\u202f°C. But, RsBBI completely lost its TI and CI activities on reduction with 3\u202fmM DTT. Conversely, RsKI lost its TI activity on heating at 100\u202f°C and retained >60% of its TI activity in presence of 3\u202fmM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils\u202f>\u202fβ-sheets/β-turns\u202f>\u202fα-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75\u202f°C. Further, the significant inhibitory activity of RsBBI (IC50\u202f=\u202f24\u202fng) and RsKI (IC50\u202f=\u202f59\u202fng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.