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

Phytochemistry. 2019 Mar:159:159-171. doi: 10.1016/j.phytochem.2018.12.018. Epub 2019 Jan 9.

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 Da) and RsKI (19,412 Da) 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 = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) 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 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) 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.

Keywords: Achaea janata (Noctuidae); Helicoverpa armigera (Noctuidae); Protease inhibitor; Rhynchosia sublobata (Fabaceae); Sodium acetate; Trichloroacetic acid; Trypsin-like midgut proteases.

MeSH terms

  • Amino Acid Sequence
  • Cajanus / embryology*
  • Chromatography, Liquid / methods
  • Dithiothreitol / chemistry
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Fabaceae / chemistry
  • Fabaceae / embryology*
  • Hot Temperature
  • Kinetics
  • Mass Spectrometry / methods
  • Protein Structure, Secondary
  • Seeds / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin Inhibitor, Bowman-Birk Soybean / chemistry*
  • Trypsin Inhibitor, Bowman-Birk Soybean / isolation & purification*
  • Trypsin Inhibitor, Kunitz Soybean / chemistry*
  • Trypsin Inhibitor, Kunitz Soybean / isolation & purification*

Substances

  • Trypsin Inhibitor, Bowman-Birk Soybean
  • Trypsin Inhibitor, Kunitz Soybean
  • Dithiothreitol