Gill S C, von Hippel P H
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Anal Biochem. 1989 Nov 1;182(2):319-26. doi: 10.1016/0003-2697(89)90602-7.
Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.
对溶液中蛋白质 - 蛋白质和蛋白质 - 配体相互作用进行定量研究需要准确测定蛋白质浓度。通常，对于仅以“分子生物学量”可得的蛋白质，很难甚至不可能对其摩尔消光系数进行准确的实验测量。然而，如果没有该参数的可靠值，就无法通过常规的紫外光谱法测定蛋白质浓度。幸运的是，通常可以通过DNA序列获得氨基酸残基序列和蛋白质分子量（进而也能知道氨基酸组成）的信息，对于大多数此类蛋白质，DNA序列通常是准确已知的。在本文中，我们提出了一种仅根据氨基酸组成来计算蛋白质在280 nm处准确的（大多数情况下精确到±5%）摩尔消光系数的方法。该方法以18种摩尔消光系数准确已知的“正常”球状蛋白质为校准对象，并讨论了该方法的基本假设及其局限性。
