Wiechelman K J, Braun R D, Fitzpatrick J D
Department of Chemistry, University of Southwestern Louisiana, Lafayette 70504.
Anal Biochem. 1988 Nov 15;175(1):231-7. doi: 10.1016/0003-2697(88)90383-1.
The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.
Cu⁺与二喹啉甲酸形成的有色络合物是二喹啉甲酸蛋白质测定法的基础（P.K.史密斯、R.I.克罗恩、G.T.赫尔曼森、A.K.马利亚、F.H.加特纳、M.D.普罗文扎诺、E.K.藤本、N.M.戈克、B.J.奥尔森和D.C.克伦克（1985年）《分析生物化学》150卷，76 - 85页）。研究表明，半胱氨酸、色氨酸、酪氨酸和肽键能够将Cu²⁺还原为Cu⁺。电化学研究以及在37℃进行反应时观察到的颜色变化程度表明，色氨酸、酪氨酸和肽键在该温度下并未完全被氧化。当反应温度升至60℃时，这三组观察到的颜色形成明显更多。对二肽、三肽、四肽以及蛋白质的研究表明，颜色形成的程度并非各个产生颜色的官能团贡献的总和。具有与半胱氨酸、胱氨酸、酪氨酸或色氨酸类似官能团的化合物被证明会与二喹啉甲酸试剂发生反应。在牛血清白蛋白存在下，这些化合物形成的颜色无法通过使用含有相同浓度干扰化合物的试剂空白来补偿。
