双缩脲反应.doc

双缩脲反应biuret reaction 蛋白质在碱性溶液中与硫酸铜作用形成紫色络合物的呈色反应。在540nm 波长处有最大吸收。可用于蛋白质的定性和定量检测。 紫色络合物分子结构式在碱性溶液（NaOH）中，双缩脲（H2NOCNHCONH2）能与铜离子（Cu2+）作用，形成紫色络合物（该物质的分子结构式见图），该反应即双缩脲反应。 双缩脲反应是肽和蛋白质所特有的，而为氨基酸所没有的一种颜色反应。一般分子中含有两个氨基甲酪基（即肽键：-CO-NH-）的化合物与碱性铜溶液作用，就会形成紫色或蓝紫色络合物。 注：除-CO-NH-有此反应外，（-CONH2-）、（-CH2-）、（-NH2-）、（-CS-CS-NH2）等基团亦有此反应。 双缩脲反应的鉴定由于蛋白质分子中含有很多与双缩脲结构相似的肽键，因此也能与铜离子在碱性溶液中发生双缩脲反应，且颜色深浅与蛋白质的含量的关系在一定范围内符合比尔定律，而与蛋白质的氨基酸组成及分子量无关，故可用双缩脲法测定蛋白质的含量（借助分光光度计可减小误差）。 双缩脲反应主要涉及肽键，因此受蛋白质特异性影响较小。使用试剂价廉易得，操作简便，可测定的范围为110mg蛋白质，适于精度要求不太高的蛋白质含量的测定，能测出的蛋白质含量须在约0.5mg以上。双缩脲法的缺点是精确度低、所需样品量大。干扰此测定的物质包括在性质上是氨基酸或肽的缓冲液，如Tris缓冲液，因为它们产生阳性呈色反应，铜离子也容易被还原，有时出现红色沉淀。 配制双缩脲试剂的注意事项双缩脲试剂由NaOH溶液（0.1g/mL）和CuSO4溶液（0.01g/mL）配制而成，配制比例为5:1。但是双缩脲试剂不用现配现用，这是与斐林试剂不同的地方之一！Biuret testThe characteristic color of a positive biuret testThe biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms a violet-colored complex in an alkaline solution.1 Several variants on the test have been developed.The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.In spite of its name, the reagent does not in fact contain biuret (H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide bonds in the biuret molecule.edit ProcedureAn aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5160mg/ml can be determined.edit Biuret reagentThe biuret reagent is made of potassium hydroxide (KOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.2Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein assay, a colorimetric assay used to determine protein concentrationsuch as UV-VIS at wavelength 540nm (to detect the Cu2+ ion).Increasing the sensitivity of the biuret testCu+ is a strong reducing agent which can react for example with Mo(VI) in Folin-Ciocalteus reagent to form molybdenum blue. In this way, proteins can be detected in concentrations between 0.005 and 2mg/mL.3 Molybdenum blue in turn can bind certain organic dyes (malachite green, Auramin O), resulting in further amplification of the signal.4Cu+ forms a deep purple complex with bicinchoninic acid (BCA)5, which allows prot