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Paper Details

Detection of Helicoverpa armigera Gut In- and Active Proteinases by In-gel Activation on Two-dimensional Gel Electrophoresis X-ray Film Contact Print Method

Vinod D. Parde*, Tewodros Aregai, Alemayehu Dubale, Mohana Rao Abburi and H. K. R. Prasad Saripalli

Journal Title:Journal of Chemical, Biological and physical sciences

Protease inhibitors (PIs) play an important role in host plant resistance to insect pests. Therefore, the present studies were carried out plant and chemical protease inhibitor for inhibition of zymogen activation, and their biological activity against the pod borer, Helicoverpa armigera (Hbner), the most important pest of agriculture and horticulture crops worldwide. The present studies indicated that H. armigera gut proteinases were synthesized in inactive isoforms, and get activated by trypsinlike proteinases. Nine proteinase bands were detected using in-gel activation on two-dimensional gel X-ray film contact print technique, and in addition to that in-gel activation method used for detection of pro-proteinase, however four activated proteinase detected when larvae fed on chemical inhibitors and three pro-proteinases bands detected on X-ray film when larvae fed on non-host plant protease inhibitors. Gelatin coating present on the X-ray film was used as a substrate to detect electrophoretically separated pro- and proteinases of H. armigera gut extract on SDS-PAGE and twodimensional gels. In gel activation method involved electrophoresis, followed by washing the gel with non-ionic detergent in case of SDS-PAGE, equilibration of the gel in trypsin (0.0001%) buffer, overlaying the gel on X-ray film, followed by washing the film with hot water to remove hydrolyzed gelatin, revealing activated pro-proteinase bands. This in-gel activation on two-dimensional gel X-ray film contact print technique is having significant application for the detection, identification and characterization of different proteolytic enzymes.