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Biophys J, April 2000, p. 2116-2126, Vol. 78, No. 4
and
*Section of Molecular and Cellular Biology, University of
California, Davis, Davis, California, Department of
Chemistry, State University of New York, Buffalo, New York,
Department of Anatomy, Physiology, and Cell Biology,
School of Veterinary Medicine, University of California, Davis, Davis,
California, USA
The effect of temperature on the binding equilibria of
calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes
that have been largely ignored. We conducted a systematic study of
thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green
dextran, Indo-1, and Fura-2 all show temperature-dependent effects on
fluorescence in all or part of the range tested (5-40°C). Specifically, the intensity of the single-wavelength dyes increased at
low temperature. The ratiometric dyes, because of variable effects at
the two wavelengths, showed, in general, a reduction in the
fluorescence ratio as temperature decreased. Changes in viscosity, pH,
oxygen quenching, or fluorescence maxima could not fully explain the
effects of temperature on fluorescence. The excited-state lifetimes of
the dyes were determined, in both the presence and absence of calcium,
using multifrequency phase-modulation fluorimetry. In most cases, low
temperature led to prolonged fluorescence lifetimes. The increase in
lifetimes at reduced temperature is probably largely responsible for
the effects of temperature on the physical properties of the
calcium-sensing dyes. Clearly, these temperature effects can influence
reported calcium concentrations and must therefore be taken into
consideration during any investigation involving variable temperatures.
Biophys J, April 2000, p. 2116-2126, Vol. 78, No. 4
© 2000 by the Biophysical Society 0006-3495/00/04/2116/11 $2.00
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