PROTEIN DETERMINATION USING BICINCHONINIC ACID IN THE PRESENCE OF SULFHYDRYL-REAGENTS
PROTEIN DETERMINATION USING BICINCHONINIC ACID IN THE PRESENCE OF SULFHYDRYL-REAGENTS
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DOI：
10.1016/0003-2697(88)90109-1
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发表时间：
1988-04-01
影响因子：
2.9
通讯作者：
STRAKA, JG
中科院分区：
文献类型：
--
作者：
HILL, HD;STRAKA, JG
发现用于蛋白质定量的二喹啉甲酸（BCA）铜试剂与硫醇试剂以线性和可重复的方式反应。与硫醇的反应性与Cu（I）-BCA络合物[6.6 × 104]测定的消光系数密切匹配。103升（摩尔Cu. cm）-1]，表明反应是定量的。该反应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蛋白试剂之前，用碘乙酰胺预孵育蛋白溶液。碘乙酰胺本身不与BCA试剂反应。在相对于硫醇当量10倍摩尔过量的碘乙酰胺的存在下，硫醇与BCA试剂的反应被阻止。该方法是简单的，并允许已通过添加硫醇试剂稳定的蛋白质溶液的测定。
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 .times. 103 liters (mol Cu .cntdot. 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.