NUSNNI-NanoCore, National University of Singapore, T-Lab Level 11, 5A Engineering Drive 1, Singapore 117580, Singapore.
Biochem Biophys Res Commun. 2011 Jul 29;411(2):455-7. doi: 10.1016/j.bbrc.2011.06.187. Epub 2011 Jul 6.
We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.
我们在此报告 N-羟基琥珀酰亚胺（NHS）在双缩脲（BCA）蛋白测定中存在严重干扰。NHS 是生物分析科学中最常用的交联剂之一，如果存在于蛋白质分析物溶液中，它可能会导致 BCA 蛋白测定中的严重潜在错误，从而导致基于 BCA 的蛋白质估计。它被鉴定为一种还原物质，通过还原 BCA 工作试剂中的 Cu(2+) 来干扰 BCA 蛋白测定。NHS 的吸光度峰值和吸光度信号与牛血清白蛋白（BSA）非常相似，从而表明 NHS 和蛋白质具有相似的 BCA 反应机制。然而，NHS 和 BSA 的组合吸光度不是加和的。BCA 蛋白测定的时间响应测量显示 NHS 的单相位动力学一致，而 BSA 的动力学逐渐降低。通过绘制 NHS 固定浓度的 BSA 的额外 BCA 标准曲线，可以有效地抵消 NHS 存在时对蛋白质估计的误差。BSA 和 NHS 固定浓度的 BSA 的吸光度值之间的差异提供了 NHS 贡献的吸光度，然后从分析物样品的总吸光度中减去 NHS 的吸光度，以确定分析物样品中蛋白质的实际吸光度。
