Quantitation of aromatic residues in proteins: model compounds for second-derivative spectroscopy.
Quantitation of aromatic residues in proteins: model compounds for second-derivative spectroscopy.
复制标题
蛋白质中芳香族残基的定量：二阶导数光谱学的模型化合物。
作者：
R. Levine;M. Federici
蛋白质在6 M胍中的紫外吸收光谱约为芳香族氨基酸、苯丙氨酸、酪氨酸和色氨酸的光谱之和，加上光散射和二硫化物的贡献。光谱的多组分分析理论上允许同时定量蛋白质中的每种芳香族氨基酸。在实践中，这是不可能的，因为氨基酸光谱的相似性，摩尔吸光系数的巨大差异，二硫化物的可变吸收，光散射，以及当氨基酸掺入蛋白质时发生的波长偏移。我们描述了一种同时定量纯化蛋白质中芳香族氨基酸的方法。我们使用二阶导数紫外光谱加上统计加权多组分分析。二阶导数的使用实际上消除了光散射和胱氨酸的干扰。模型化合物的经验选择避免了波长偏移的问题。模型是N-乙酰苯丙氨酸乙酯在6 M胍中的苯丙氨酸，N-乙酰酪氨酸乙酯在55%甲醇中的酪氨酸，和蜂毒素在6 M胍中的色氨酸。该方法允许准确，快速定量的苯丙氨酸，酪氨酸，色氨酸在完整的，变性的蛋白质。
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.