Photophysics of Aqueous Tryptophan: pH and Temperature Effects

The fluorescence decay kinetics of aqueous tryptophan and 3-methylindole have been determined as a function of pH and temperature by using a picosecond dye laser-single photon counting system with a time resolution of 50 ps. At pH 11, tryptophan exhibits a single exponential decay, with a lifetime of 9.1 ns at 18 O C . However, at pH 7 the decay is faster and definitely nonexponential; the values obtained from a biexponential fit to the data at pH 7 are T ] = 0.43 ns, 7 = 3.32 ns, a n d f = 0.19 at 18 O C . The behavior of a 3-methylindole closely resembles that of tryptophan at pH 11. A model for the photophysics of aqueous tryptophan is presented in which the excited-state decay constant at pH 11 (where the amino acid side chain is not protonated) is given by the superposition of three independent processes: fluorescence, intersystem crossing, and photoionization; of these processes only photoionization is temperature sensitive (E' = 51 kJ mol-'). In the region pH 4-8, where tryptophan exists in the zwitteridn form, a new nonradiative process is introduced, which involves intramolecular proton transfer from the -NH3+ group to the excited indole ring. The apparent activation energy for intramolecular quenching (E' = 16 kJ mol-') suggests that it is a predominantly diffusion-controlled process. It is proposed that the nonexponential decay observed for aqueous tryptophan at pH 7 arises from transient terms in the rate constant for intramolecular quenching. Quantum yields calculated from this model compare well with experimental values.