Frequency-domain fluorometry was used to investigate the time-dependent intensity decays of N-acetyl-L-tryptophanamide (NATA) when collisionally quenched by acrylamide and iodide in propylene glycol at 20°C. The intensity decays of NATA became increasingly heterogeneous in the presence of quenching, and the steady-state Stern-Volmer plots show significant upward curvature. These frequency-domain and steady-state data clearly indicate that the rate constants for quenching of NATA by acrylamide and iodide depend exponentially on the fluorophore-quencher separation distance, The NATA intensity decays were not consistent with the Smoluchowski-Collins-Kimball radiation boundary condition for quenching. The upward curvature of the Stern-Volmer plots provide a sensitive method to determine the characteristic distances for fluorophore-quencher interaction. The rate constant for quenching are discussed based on the mechanism of interaction between tryptophanyl fluorophore and quencher molecules which appear to involve electron transfer for acrylamide and heavy atom effect for iodide. These results suggest that the distance-dependent interaction and the effects of solvent dynamics need to be considered in the interpretations of data for quenching of proteins.
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