Quantitation of aromatic residues in proteins: model compounds for second-derivative spectroscopy

The ultraviolet absorption spectrum of proteins in 6 M guanidine is approximately that of the sum of the spectra of the constituent aromatic amino acids, phenylalanine, tyrosine, and tryptophan, plus contributions from light scattering and disulfides. A multicomponent analysis of the spectrum would theoretically permit simultaneous quantitation of each aromatic amino acid in the protein. In practice, this has not been possible, because of the similarities of the spectra of the amino acids, large differences in molar absorptivity, variable absorption by the disulfides, light scattering, and wavelength shifts which occur when the amino acids are incorporated into proteins. We describe a method for the simultaneous quantitation of the aromatic amino acids in purified proteins. We used second-derivative ultraviolet spectroscopy coupled with a statistically weighted multicomponent analysis. Use of the second derivative virtually eliminated interference from light scattering and from cystine. Empirical selection of model compounds obviated the problem of wavelength shifts. The models are N-acetylphenylalanine ethyl ester in 6 M guanidine for phenylalanine, N-acetyltyrosine ethyl ester in 55% methanol for tyrosine, and mellitin in 6 M guanidine for tryptophan. This method permits accurate, rapid quantitation of phenylalanine, tyrosine, and tryptophan in intact, denatured proteins.