BASIC THEORY AND INTERPRETATION OF FLUORESCENCE SPECTRA When radiation is incident on a solution, most of it is scattered without change in wavelength.

Thus, exciting radiation at 280 nm will be scattered and a fraction of it will be detected by the spectrofluorometer as a peak at the same wavelength. The width of this Rayleigh scattering peak (Fig. B3.6.1) will depend on instrumental settings, mainly those of the excitation and emission slit widths, which determine the range of wavelengths observed. The bandwidth is the width, in nm, of the distribution of intensity measured at half the height of the maximum intensity. A further low-intensity peak, known as the Raman band, is also observed in spectra (see Fig. B3.6.1). This is a low-intensity band of scattered radiation whose distance from the excitation band is a measure of the vibrational energy of the HO bond in solvent water. At ex = 280 nm, the Raman band for water occurs at 31 1 nm; in general, it occurs at 1/{(1/ex) (3.6 104)}, where exis the excitation wavelength in nm. The intensity and resolution of the Raman band provide a useful empirical check on the performance of the spectrofluorometer. A decrease in the signal-to-noise ratio usually indicates a deterioration of the lamp. Chromophores, such as the sidechains of tryptophan and tyrosine, will absorb some of the radiation in 1015 sec and become excited from the ground electronic state (S0) tohigher vibrational levels in a higher energy electronic state (S1; see Fig. B3.6.7). The energy in the vibrational levels is rapidly lost (1012 sec) to the surroundings as thermal energy, so that the excited chromophore is very largely in its ground vibrational level.

Return to the ground electronic state can result in the emission of radiation of lower energy and hence longer wavelength (Figs. B3.6.1 and B3.6.3), or the energy can be dissipated in a number of ways that are nonradiative (Fig. B3.6.7), including quenching (see Quenching, below). The ratio of the rate constants for these two alternative processes defines the quantum yield or fluorescence efficiency, which is almost always less than unity. The chromophores tyrosine and tryptophan absorb radiation in the 280 nm region, re-emitting a proportion of it as fluorescence. The quantum yields for tryptophan, tyrosine, and phenylalanine are 0.2, 0.1, and 0.04, respectively. Coupled with their relative absorption coefficients (UNIT B1.3), it can be seen why most protein fluorescence spectra are dominated by tryptophan. Quantitation of the fluorescent properties of a protein can provide a number of rather specific parameters that allow assessment of the correct folding, besides giving additional structural information.