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

Mohanraj, S S and Gujjarlapudi, M and Lokya, V and Mallikarjuna, N and Dutta-Gupta, A and Padmasree, K (2019) Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea. Phytochemistry (TSI), 159. pp. 159-171. ISSN 00319422

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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.

Item Type: Article
Divisions: Research Program : Genetic Gains
CRP: UNSPECIFIED
Uncontrolled Keywords: Achaea janata (Noctuidae), Helicoverpa armigera (Noctuidae), Rhynchosia sublobata (Fabaceae), Protease inhibitor, Trypsin-like midgut proteases, Trichloroacetic acid, Sodium acetate, pigeonpea
Subjects: Others > Helicoverpa
Mandate crops > Pigeonpea
Others > Genetics and Genomics
Depositing User: Mr Ramesh K
Date Deposited: 12 Mar 2019 09:33
Last Modified: 12 Mar 2019 09:33
URI: http://oar.icrisat.org/id/eprint/11087
Official URL: http://dx.doi.org/10.1016/j.phytochem.2018.12.018
Projects: UNSPECIFIED
Funders: Department of Biotechnology (DBT), New Delhi, India
Acknowledgement: This work was supported by grants from Department of Biotechnology (DBT), New Delhi, India (Ref.BT/PR13261/AGR/05/ 489/2009/(letter I) dt. 21/12/2010) to KPS (PI) and ADG (Co-PI). SSM is thankful to UoH for BBL fellowship and DBT-Project for JRF and SRF. We thank Prof. A. S. Raghavendra for extending his lab facilities. We thank Prof. Sarada. D. Tetali for her critical suggestions during execution of project and providing lab facilities. We thank Ms. Monika Kannan from Proteomics facility, SLS, UoH, and Dr. Madhura Rekha, Ms. Hima Bindu and Mr. Joy Prasanth from Sandor Proteomics, Hyderabad, Telangana, India for their help in MALDI-TOF studies.
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