J. Biol. Chem., Vol. 264, Issue 26, 15392-15397, 09, 1989
Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects
ST Ferreira and S Verjovski-Almeida
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
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.
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