Mach H, Middaugh C R, Lewis R V
Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Anal Biochem. 1992 Jan;200(1):74-80. doi: 10.1016/0003-2697(92)90279-g.
Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.
蛋白质浓度的光谱测量需要知道相关消光系数的值。然而，如果蛋白质的氨基酸组成已知，只要知道蛋白质中色氨酸和酪氨酸残基的摩尔吸光率值，就可以近似计算消光系数。我们应用了矩阵线性回归程序以及实验值与计算值之间平均绝对偏差的映射，以找到色氨酸的摩尔消光系数（εM，1 cm，280 nm）为5540 M-1 cm-1，酪氨酸残基的摩尔消光系数为1480 M-1 cm-1，这是由一组超过30种蛋白质的实验确定的消光系数所定义的“平均”蛋白质。与从盐酸胍中的模型化合物溶液获得的常用值相比，使用这些值可显著改进消光系数的估计。与通常在实验确定的消光系数之间观察到的大偏差相比，这些结果的一致性表明，该方法在球状蛋白质摩尔吸光率的初始估计中可能提供可接受的准确性。
