Mechanism of quenching of N-acetyl-tryptophan-amide fluorescence by imidazole

The fluorescence lifetime of N-acetyl-tryptophan-amide (NATA) was measured by multifrequency phase fluorometry, in the presence of increasing concentrations of imidazole. Two pH values were tested, pH 4.5 where imidazole is fully protonated and pH 9.0 where it is fully unprotonated. At both pH values, the inverse lifetime increases in a nonlinear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analyzed in terms of the formation of a complex with a reduced fluorescence lifetime. The rate constants for association (at 25 degree(s)C) are around 5 (+/- 0.2) X 109 M-1 s-1 and are thus diffusion controlled. The association equilibrium constant is strongly pH-dependent and is much higher than the expected value of 0.4 M-1 for a collisional complex. The intrinsic fluorescence lifetime of the complex is 1.56 (+/- 0.02) ns at pH 9.0 and 1.82 (+/- 0.03) ns at pH 4.5, as compared to 2.37 (+/- 0.03) ns for free tryptophan at pH 9.0 and 2.83 (+/- 0.05) at pH 4.5 (all at I equals 0.34). This means that at both pH values the fluorescence lifetime of tryptophan in the complex is reduced to 61% (+/- 0.05) of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, due to a longer residence-time of the complex. At high viscosity the association equilibration is too slow and the system is described by two lifetimes. The quenching effect of His-18 on the fluorescence of the proximal Trp-94 of Barnase is discussed in terms of these findings. An extensive account of these results has appeared.