Resonance Raman study of the chromophore-specific vibrational and electronic of bilirubin

Abstract

UV–VIS absorption and resonance Raman (RR) studies have been used to elucidate the chromophore-specific electronic and vibrational spectroscopic bands of bilirubin in chloroform and in aqueous solution at pH 10 and 7.4. RR excitation profiles reveal two sets of chromophore-specific bands in each case, arising from the AB and CD pyrromethenone chromophores. A band-shape analysis of the UV–VIS absorption spectra in combination with these excitation profiles provides the absorption maxima of the individual chromophores. In the neutral species which is present in chloroform, the AB chromophore absorbs at a longer wavelength than the CD chromophore. However, in alkaline aqueous solution, where the dianion is formed, the AB chromophore absorbs at the shorter wavelength as a consequence of a large blue shift of this band. In pH 7.4 aqueous solution, the AB chromophore absorbs at the longer wavelength owing to the formation of a monoanion in which the strong internal hydrogen bonding of the lactam A group is retained, while the proton which is hydrogen bonded to the lactam D group is removed. The RR spectra support this interpretation.