Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria
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Mohammad S. Akbar, Kuruba Sreeramulu, Hari C. Sharma. (30/6/2016). Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria. Journal of Bioenergetics and Biomembranes, 48 (3), pp. 241-7.
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. Methylparathion,
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.