Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects.
Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.
Ferreira
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Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Verjovski-Almeida
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates; D013997:Time Factors; D014364:Tryptophan
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D013861:Thiocyanates; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D002118:Calcium