Slocum T L, Deupree J D
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6260.
Anal Biochem. 1991 May 15;195(1):14-7. doi: 10.1016/0003-2697(91)90287-4.
The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.
使用二辛可宁酸（BCA）测量蛋白质浓度已被广泛接受，因为该试剂对许多与各种组织和酶制剂一起使用的缓冲液、蔗糖溶液和去污剂不敏感。然而，任何能够在碱性介质中还原Cu2+的化合物，如生物胺，都会产生颜色反应。本研究的主要目的是确定神经组织中存在的生物胺是否会干扰使用BCA方法测量蛋白质。发现在1至100 nmol/2.1 ml测定体积的浓度范围内，儿茶酚胺会使BCA试剂的颜色呈线性增加。儿茶酚胺似乎比血清素或抗坏血酸对BCA试剂更敏感。蛋白质浓度为50 nmol/mg或测定体积为1 nmol/2.1 ml或更高时的儿茶酚胺，会产生比单独蛋白质明显更高（P小于0.0001）的颜色反应。BCA试剂对于测量完整突触小泡和嗜铬颗粒的蛋白质浓度并不理想，因为这些细胞器中的儿茶酚胺浓度高到足以使产生的颜色比单独蛋白质观察到的颜色增加1.1至2.5倍。儿茶酚胺产生的颜色发展的线性表明，BCA可用于定量1至100 nmol之间的儿茶酚胺浓度。BCA试剂无法区分不同的儿茶酚胺。
