[Materials and Methods] [Time Course] [Title page]
Results and Discussion: Quenching of SPQ fluorescence
SPQ fluorescence is quenched
by halides via a collisional mechanism (8). To examine the fluorescence
quenching in our system, the fluorescence intensity of SPQ was
measured as a function of chloride concentration. The data are
plotted in Figure 1 as a Stern-Volmer plot. The linearity of the
plot indicates that SPQ fluorescence quenching in our system is
described reasonably well by a collisional quenching mechanism
with an apparent quenching constant 0.071 ± 0.016 mM-1. Lifetime
measurements also support a collisional quenching mechanism. In
the absence of chloride, SPQ has a fluorescence lifetime of 26.0 ±
0.5 ns which decreases to 2.3 ± 0.1 ns in the presence of (75 mM
chloride + 75 mM bicarbonate). The fractional quenching predicted
from these lifetime measurements is 0.09, while that measured
from equilibrium measurements at 75 mM chloride is 0.16. This
comparison indicates that the quenching mechanism is more complex
than a simple bimolecular collisional mechanism.
|
| Figure 1. Dynamic quenching of SPQ by chloride and bicarbonate solutions. The fluorescence of a solution of 500 [micro]M SPQ, 20 mM HEPES, pH 7.4, and varying chloride and bicarbonate concentrations (total anion concentration = 150 mM) was measured at 25 °C. The data represent the average of three experiments. A weighted fit of a linear function to this data gives an apparent quenching constant of 0.064 ± 0.004 mM-1. Four sets of these experiments gave an average quenching constant of 0.071 ± 0.016 mM-1. Inset: Dynamic quenching of SPQ fluorescence by HEPES.
|
Our value of 0.071 mM-1 for the quenching constant differs significantly
from the value of 0.118 mM-1 reported previously (8). Our experiments
were done in the presence of three ions: chloride, bicarbonate
and HEPES. To assess the effect of the individual ions, we measured
the fluorescence intensity of SPQ as a function of the concentration
of these ions separately. For chloride alone, we obtained a quenching
constant of 0.116 0.007 mM-1 (data not shown), similar to that
reported previously (8). Bicarbonate at 150 mM had no detectable
effect on SPQ fluorescence. HEPES quenched SPQ fluorescence with
a quenching constant of 0.03 0.003 mM-1 (inset, Figure 1). The
apparent quenching constant for chloride in the presence of 20
mM HEPES and bicarbonate, calculated from equation 3, is 0.072
mM-1. Since this calculated value is equal to our measured quenching
constant, the difference between our quenching constant and that
determined previously is due to the quenching of SPQ fluorescence
by HEPES.
[Materials and Methods] [Time Course] [Title page]