The accessibility of fluorescent groups in proteins can be determined with charged quenching agents which provide information about conformational topography and local charge distribution surrounding the fluorescent moiety. Micelles of sodium dodecyl sulfate and cetyltrimethylammonium bromide containing anthracene were used to characterize the quenching properties of pyridinium and iodide ions in a model system where the fluorophore and charge distribution are known with certainty. Iodide ions were effective quenchers only with positively charged cetyltrimethylammonium bromide micelles. Pyridinium ions quenched anthracene fluorescence only in negatively charged sodium dodecyl sulfate micelles. The quenching constants for pyridinium chloride in ethanol and sodium dodecyl sulfate micelles were 42 and 520 M-1, respectively. For potassium iodide the corresponding values in ethanol and cetyltrimethylammonium bromide micelles were 17 and 418 M-1. When cetylpyridinium ions were incorporated into the cetyltrimethylammonium bromide micellar structure the quenching constant was 348 M-1. If the micelle-anthracene systems interacted with an ion of opposite charge, the quenching constant, Ksv, was about ten times greater than that predicted by diffusion controlled kinetics. If the quencher and the micelle-anthracene system had like charges, Ksv was practically nil. These results establish that iodide and pyridinium ions exhibit vastly different quenching capacities which are dependent on local charge distribution. Adequate interpretation of quenching studies with biological systems such as proteins, lipoproteins, and membranes where the microscopic environment is unknown can be more adequately interpreted if both probes are employed to obtain the necessary complementary information.
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