Atherton B A, Cunningham E L, Splittgerber A G
Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, USA.
Anal Biochem. 1996 Jan 15;233(2):160-8. doi: 10.1006/abio.1996.0024.
The Coomassie brilliant blue dye-binding method for protein assay has become important relatively recently. The basis of the assay method is the binding of dye to protein, with production of a dye-protein complex which absorbs light intensely at 620 nm, but the mechanism of the binding process is not well understood. In this paper, two mathematical models for the binding process are developed, one involving the binding of both protonated (green) and deprotonated (blue) forms of the dye. The second model allows only binding of the blue species to proteins. These models are tested for their ability to estimate number of dye-binding sites (n) and binding constants (Kd) from protein assay data. The models are also tested for their ability to reproduce the experimental assay curve using either known values or reasonable estimates of the equation parameters. The models are shown to be approximately equal in ability to reproduce experimental data related to the protein assay, which somewhat favors the simpler of the two models. In this paper, a method for estimating n and Kd from standard curve-fitting procedures is established. Hitherto, binding constants were only available from assay data taken under conditions of very large molar protein/dye ratios. The possibility of protonated forms of the dye binding to proteins was not ruled out by this study, but for many purposes the use of the simple dye-binding model, in which only the deprotonated dye species binds, is sufficient.
考马斯亮蓝染料结合法用于蛋白质测定是相对较新才变得重要起来的。该测定方法的基础是染料与蛋白质的结合，形成一种在620nm处有强烈吸光能力的染料 - 蛋白质复合物，但结合过程的机制尚未完全清楚。在本文中，针对结合过程开发了两种数学模型，一种涉及染料的质子化（绿色）形式和去质子化（蓝色）形式的结合。第二种模型只允许蓝色形式的染料与蛋白质结合。对这些模型进行了测试，看它们从蛋白质测定数据中估计染料结合位点数量（n）和结合常数（Kd）的能力。还测试了这些模型使用方程参数的已知值或合理估计值来重现实验测定曲线的能力。结果表明，在重现与蛋白质测定相关的实验数据方面，这些模型的能力大致相当，这在一定程度上支持了两种模型中较简单的那个。本文建立了一种从标准曲线拟合程序中估计n和Kd的方法。在此之前，结合常数只能从在非常大的蛋白质/染料摩尔比条件下获得的测定数据中得到。本研究并未排除染料的质子化形式与蛋白质结合的可能性，但在许多情况下，使用仅去质子化染料物种结合的简单染料结合模型就足够了。
