Drochioiu G, Damoc N E, Przybylski M
Faculty of Chemistry, "Al. I. Cuza" University of Iasi, 11 Carol I, Iasi 700506, Romania.
Talanta. 2006 May 15;69(3):556-64. doi: 10.1016/j.talanta.2005.10.026.
A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240nm. Copper absorbance slowly increases from 240 to 190nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu(2+) ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu(2+) ions at pH 10.3. To determine proteins, 1-1.5ml of 1.8% KOH solution with 0-20mugml(-1) protein is treated with 25mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226nm in 1cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20mugml(-1) protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.
已开发出一种基于紫外区双缩脲吸收的非常简单、高选择性且灵敏的蛋白质检测方法。著名的双缩脲检测法是基于蛋白质在强碱性条件下与铜离子反应形成铜 - 蛋白质复合物。然而，如果在紫外范围内进行测量，铜离子可能会严重干扰测定。在本方法中，蛋白质在不同pH值下从不溶性盐中 mobilize 铜离子，并通过紫外分光光度法和质谱法研究铜 - 蛋白质复合物。使用磷酸铜时，游离铜离子在540至240nm范围内不干扰测定。铜的吸光度从240nm缓慢增加到190nm，在此处建议使用试剂空白。在226nm和高pH值下发现铜 - 蛋白质复合物的最大吸光度。用质谱仪直接测量的铜 - 蛋白质复合物的化学计量比取决于pH值：在pH 7.4时，没有任何组氨酸残基的肽的一半仅螯合单个Cu(2+)离子，但在pH 10.3时，每个这样的肽可 mobilize 1至6个Cu(2+)离子。为了测定蛋白质，将1 - 1.5ml含0 - 20μgml(-1)蛋白质的1.8% KOH溶液与25mg磷酸铜粉末混合。将混合物剧烈搅拌、离心，然后在1cm石英比色皿中以试剂空白为对照，在226nm处测量上清液的吸光度。在此波长下，该颜色体系在0.1 - 20μgml(-1)蛋白质范围内符合比尔定律。摩尔吸光系数值被证明是每种被分析蛋白质的特征。因此，应使用单个蛋白质绘制校准曲线。该检测方法被证明比经典双缩脲法灵敏100倍以上。该方法具有高度选择性，测定几乎不受其他物质存在的影响。研究了所有其他重要的分析参数，并通过对一些生物材料的分析验证了该方法的实际适用性。 （注：原文中“mobilize”一词在生物学语境中可能是“动员、促使……移动、使……释放等”意思，这里暂保留英文未翻译，因为不确定准确的中文释义，需结合更专业知识进一步确定其准确含义）
