Hill H D, Straka J G
Center for the Study of Advanced Liver Disease, University of Minnesota, Minneapolis 55455.
Anal Biochem. 1988 Apr;170(1):203-8. doi: 10.1016/0003-2697(88)90109-1.
The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.
用于蛋白质定量的二喹啉甲酸（BCA）铜试剂被发现能与硫醇试剂以线性且可重复的方式发生反应。其与硫醇的反应活性与为Cu（I）-BCA络合物测定的消光系数[6.6×10³升/（摩尔铜·厘米）⁻¹]紧密匹配，这表明该反应是定量的。此反应会干扰蛋白质浓度的准确测定。开发了一种使用BCA蛋白质试剂在硫醇试剂存在下测定蛋白质浓度的方法。该程序包括在加入BCA蛋白质试剂之前，将蛋白质溶液与碘乙酰胺预孵育。碘乙酰胺本身不与BCA试剂反应。当碘乙酰胺的摩尔量比硫醇当量过量10倍时，可防止硫醇与BCA试剂的反应。该方法简单，可用于检测已通过添加硫醇试剂而稳定的蛋白质溶液。
