title: Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.
abstract: Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery.
authors.affiliation: Entomology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, 502324, India.|Department of Biochemistry, Gulbarga University, Gulbarga, Karnataka, 585106, India.
authors.forename: S Md
authors.initials: SM
authors.lastname: Akbar
authors.affiliation: Department of Biochemistry, Gulbarga University, Gulbarga, Karnataka, 585106, India.
authors.forename: K
authors.initials: K
authors.lastname: Sreeramulu
authors.affiliation: Entomology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, 502324, India. h.sharma@cgiar.org.
authors.forename: Hari C
authors.initials: HC
authors.lastname: Sharma
mesh_terms: D000818:Animals; D005453:Fluorescence; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007306:Insecticides; D008024:Ligands; D008565:Membrane Proteins; D014364:Tryptophan
keywords: Cytochrome c; Insecticide; Mitochondria; Trp fluorescence quenching