Knowledge Bank

UV resonance Raman excitation profiles of phenylalanine, tyrosine, and tryptophan have been measured in the UV spectral region below 300 nm. The excitation profiles of each of these derivatives are characteristic of their resonant electronic transitions. Little enhancement is observed for excitation within the almost symmetry forbidden electronic transition at 260 nm for phenylalanine. Most of the Raman intensity derives from higher energy electronic transitions. Some enhancement is observed in the 275 nm absorption band of tyrosine. In tryptophan the excitation profiles show significant intensity and structure in the 280 nm and the 220 nm absorption bands. The excitation profile maxima of these aromatic amino acids are sufficiently separated that a judicious choice of excitation wavelength permits selective enhancement of each of these aromatic amino acids in an equimolar aqueous mixture. For example, excitation at 225 nm specficially enhances the 757, 877, 1006, 1350, 1550, and 1615 peaks of tryptophan as well as the 824, 1175, 1208,, 1517, 1556, and 1601 bands of tyrosine. Significantly less enhancement is observed for the 1000, 1190, and $1590cm-1$ Raman bands of phenylalanine. Thus, the resonance Raman spectra of an equimolar solution of the mixture excited at 225 nm is dominated by the $1006cm-1$ and $1350cm-1$ peaks of tryptophan and the $1175cm-1$ peak of tyrosine. The peaks occurring in the $1500-1700cm-1$ region are of comparable intensity and overlap. The excitation profile data detail vibronic interactions within the aromatic amino acid absorption bands. The resonance Raman spectra of the aromatic amino acids will be compared with that of simpler molecules such as benzene, phenol, phenolate and indole. The environmental dependance of the resonance Raman spectra will also be discussed. Little fluorescence interference is observed upon excitation in the 220-270 nm spectral region for tyrosinate, and the resonance Raman spectra of tryptophan can be obtained at wavelengths as long as 280 nm, well within its lowest energy singlet absorption band. These results indicate that UV resonance Raman spectroscopy can be used to probe aromatic amino acids in proteins.