Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Protein Sci. 2013 Jun;22(6):851-8. doi: 10.1002/pro.2253. Epub 2013 Apr 29.
Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.
定量研究在分子和结构生物学中通常需要准确而精确地确定蛋白质浓度，最好通过一种快速而直接的方法来实现。测量 280nm 处的紫外吸光度已被证明特别有用，因为 280nm 处的摩尔吸光率（消光系数）可以直接从蛋白质序列中预测。然而，该方法仅适用于含有色氨酸或酪氨酸残基的蛋白质。205nm 处的吸光度（以及其他波长）已被用作替代方法，尽管通常使用必须针对每种蛋白质单独校准的吸光率值，或者只能粗略估计。在这里，我们提出并验证了一种从氨基酸序列直接预测蛋白质或肽在 205nm 处的摩尔吸光率的方法，允许准确确定不含有酪氨酸或色氨酸残基的蛋白质的浓度。该方法简单易行，无需校准，适用于广泛的蛋白质和肽。
