Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds Ashok P. Giri a,1 , Abhay M. Harsulkar a,1 , Maurice S.B. Ku a , Vidya S. Gupta b , Vasanti V. Deshpande b , Prabhakar K. Ranjekar b , Vincent R. Franceschi a, * a School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA b Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India Received 2 August 2002; received in revised form 21 February 2003 Abstract Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two- dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28–6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman–Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman–Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera,a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase- like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Lepidoptera; Noctuidae; Leguminosae; Helicoverpa armigera; Psophocarpus tetragonolobus; Winged bean; Insect gut proteinases; Proteinase inhibitors; Two-dimensional gel electrophoresis; Activity visualization 1. Introduction Plants are under constant attack by insect pests, and one potent defense response by plants is to produce inhibitors against the insect’s gut proteinases (Ryan, 1973). Through evolutionary processes, insects adapt to plant proteinase inhibitors (PIs) by developing protei- nase variants that are minimally affected by the plant defense proteins (Broadway, 1995, 1997; Jongsma et al., 1996; Giri et al., 1998). In addition, it appears that some polyphagous insects are capable of altering expression of different gut proteinase isoforms to maximize their resistance to the PIs produced by a particular plant species (Michaud, 1997; Wu et al., 1997; Girard et al., 1998; Patankar et al., 2001). While there is considerable interest in the use of plant PIs for engineering resistance to insects in crop species, for some insects the currently known PIs are ineffective (Jouanin et al., 1998; Schuler at al., 1998). A useful strategy for enhancing plant defense systems in the future is to identify PIs with high activity against the particular target insect (Koiwa et al., 1998). This will likely entail the examination of plant species beyond the host group (Jongsma et al., 1996). 0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00181-X Phytochemistry 63 (2003) 523–532 www.elsevier.com/locate/phytochem * Corresponding author. Tel.: +1-509-335-3052; fax: +1-509-335- 3517. E-mail address: vfrances@mail.wsu.edu (V.R. Franceschi). 1 Present address: Plant Molecular Biology Unit, Division of Bio- chemical Sciences, National Chemical Laboratory, Pune 411 008, India.