European biophysics journal vol:20 issue:3 pages:177-182
The fluorescence life-time 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 life-time increases in a non-linear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analysed in terms of the formation of a complex with a reduced fluorescence life-time. The rate constants for association (at 25-degrees-C) are around 5 (+/- 0.2) x 10(9) 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 life-time 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 NATA at pH 9.0 and 2.83 (+/- 0.05) at pH 4.5 (all at I = 0.34). This means that at both pH values the fluorescence life-time of NATA in the complex is reduced to 61 (+/- 0.5)% of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, owing to a longer residence-time of the complex. At higher viscosity the association equilibration is too slow and the system is described by two life-times. The quenching effect of His-18 on the fluorescence of the proximal Trp-94 of barnase (Loewenthal et al. 1991, Willaert et al. 1991) is discussed in terms of these findings.