Histidine photodegradation during UV resonance Raman spectroscopy

We report photodegradation of imidazole [ImH], 4-methylimidazole [4MeImH], and histidine during collection of UV resonance Raman [UVRR] spectra. The degradation rate of histidine residues in two proteins, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and the CO adduct of hemoglobin, was also examined. A two-photon mechanism is proposed for ImH, in which photoexcitation from an initially excited state produces ImH(.+) cation radical, which deprotonates and dimerizes or reacts with other ImH molecules or with O-2. Photodegradation rates were measured for spherical and cylindrical focusing lenses, as well as for continuous wave and pulsed laser excitation. The cylindrical lenses slowed photodegradation 5-fold, while pulsed excitation (0.3 muJ, similar to20 ns, 1 kHz) speeded it up similar to2.5-fold. Histidine photodegradation is an insidious problem in protein UVRR spectroscopy because of the weakness of the histidine Raman signals. By keeping the laser power low and using cylindrical lenses, one can minimize damage to histidine while maintaining acceptable signal levels.