Sampson D L, Chng Y L, Upton Z, Hurst C P, Parker A W, Parker T J
Tissue Repair and Regeneration Program, Brisbane, QLD 4059, Australia.
Anal Biochem. 2013 Nov 1;442(1):110-7. doi: 10.1016/j.ab.2013.07.027. Epub 2013 Jul 30.
Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.
总蛋白浓度的测定是任何以蛋白质或肽为中心的分析流程中的关键步骤。本研究表明，尿胆素（一种血红素的分解产物，也是尿液的主要成分）会对二辛可宁酸（BCA）测定法产生显著干扰。这种干扰可能是由于尿胆素倾向于将铜离子（Cu(2+)）还原为亚铜离子（Cu(1+)），从而模拟了该测定法中蛋白质对铜的还原作用。此外，研究还表明，考马斯亮蓝法对尿胆素和其他小分子的影响具有更强的抗性。因此，尿胆素对通过BCA测定法获得的总蛋白浓度估计值有很强的混杂效应，所以该测定法不应用于尿蛋白定量。建议改用考马斯亮蓝法。
