Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential.
Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.
Department of Biochemistry, Institute of Biology, University of Campinas, Campinas 13083-970, SP, Brazil; Department of Food Technology and Public Health, Centre for Biological and Health Sciences, University of Mato Grosso do Sul, Campo Grande 79070-900, MS, Brazil.
Miriam
M
Dantzger
Department of Biochemistry and Molecular Biology, University of Ceara, Fortaleza 60451-970, CE, Brazil.
Ilka Maria
IM
Vasconcelos
Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, Campinas 13083-970, SP, Brazil; Institute of Biology, University of Campinas, Campinas 13083-970, SP, Brazil.
Valéria
V
Scorsato
Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, Campinas 13083-970, SP, Brazil.
Ricardo
R
Aparicio
Department of Biochemistry, Institute of Biology, University of Campinas, Campinas 13083-970, SP, Brazil.
Sergio
S
Marangoni
Department of Biochemistry, Institute of Biology, University of Campinas, Campinas 13083-970, SP, Brazil; Department of Food Technology and Public Health, Centre for Biological and Health Sciences, University of Mato Grosso do Sul, Campo Grande 79070-900, MS, Brazil. Electronic address: bioplant@terra.com.br.
Maria Lígia Rodrigues
ML
Macedo
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000032:analysis; D031292:Clitoria / Q000737:chemistry*; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D009036:Moths / Q000187:drug effects / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry*; D014357:Trypsin / Q000032:analysis
D007306:Insecticides; D011480:Protease Inhibitors; D002918:Chymotrypsin; D014357:Trypsin
Bowman–Birk proteinase inhibitor; Characterisation; Clitoria fairchildiana; Fabaceae; Lepidopteran pests; Protein purification