title: Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy.
abstract: Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.
mesh_terms: D015077:2-Hydroxy-5-nitrobenzyl Bromide; D008667:Metalloproteins / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis; D013059:Spectrum Analysis, Raman; D013774:Tetranitromethane; D014364:Tryptophan / Q000031:analogs & derivatives / Q000032:analysis*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis*
chemical_list: D008667:Metalloproteins; D010940:Plant Proteins; C003343:stellacyanin protein, plant; D014443:Tyrosine; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D013774:Tetranitromethane