Time-Resolved Protein Fluorescence

Abstract

The previous chapter described the general features of intrinsic protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties are influenced by the surrounding protein structure. We now describe time-resolved measurements of protein fluorescence. Such measurements have become increasingly common because of the increased availability of timedomain (TD) and frequency-domain (FD) instrumenta-tion.1,2 However, time-resolved studies of intrinsic protein fluorescence are made challenging by the lack of simple pulsed light sources. Pulsed laser diodes are not yet available for excitation of protein fluorescence. Pulsed LEDs for excitation of protein fluorescence have just been announced, but the pulse widths are over one nanosecond. Single-photon excitation of protein fluorescence requires wavelengths in the range from 280 to 305 nm. Prior to about 2000, the dominant pulsed light source for this range of wavelengths was a synchronously pumped cavity-dumped dye laser, typically rhodamine 6G, which was doubled to obtain the UV wavelengths. The synchronously pumped dye lasers require an actively mode-locked pump laser, typically an argon ion or Nd:YAG laser. At present the actively mode-locked lasers are becoming less available because of the widespread use of Ti:sapphire lasers. These lasers spontaneously mode lock and may not use an active mode locker. Wavelengths suitable for excitation of protein fluorescence can be obtained by frequency tripling the long-wavelength output of a Ti:sapphire laser. Tripling the output at 840 nm yields 280 nm. The femtosecond pulse widths from Ti:sapphire lasers make it practical to generate such harmonics. Excitation of intrinsic protein fluorescence can also be accomplished with synchrotron radiation. Many of the recent studies of time-resolved intrinsic protein fluorescence used the frequency-tripled output of a pulsed Ti:sap-phire laser or synchrotron radiation. Even with the best available instrumentation it is challenging to interpret the time-dependent data from proteins.