Chen Fang-Fang, Wang Shi-Long, Liu Xiang-Hu, Xu Ran, Gao Hong-Wen
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China.
Anal Chim Acta. 2007 Jul 16;596(1):55-61. doi: 10.1016/j.aca.2007.05.062. Epub 2007 Jun 8.
The interaction of brilliant red X-3B (BRX) with bovine serum albumin (BSA) in three pH media has been characterized by the spectral correction technique. The binding number maximum of BRX was determined to be 102 at pH 2.03, 82 at pH 3.25 and 38 at pH 4.35 and the binding mechanism was analyzed in detail. The effects of ionic strength from 0 to 1 mol L(-1) and temperature from 20 to 70 degrees C on the binding were investigated. The results showed that the interaction of BRX with BSA responded to the Langmuir adsorption isothermal model and the binding constant was determined. From the correlation between the binding number and the number of basic amino acid residues, the ion-pair attraction induced the union of non-covalent bonds including H-bond, van der Waals force and hydrophobic bond and the binding model was illustrated. The binding of BRX to BSA has resulted in change of the BSA conformation confirmed by means of circular dichroism. Using this interaction at pH 2.03, a sensitive method named the absorbance ratio difference spectrometry was established and applied to the protein assay and the limit of detection of protein was only 6 microg L(-1). Two samples were determined and the results were in agreement with those obtained by the classical coomassie brilliant blue colorimetry.
采用光谱校正技术研究了亮红X-3B（BRX）在三种pH介质中与牛血清白蛋白（BSA）的相互作用。测定了BRX在pH 2.03时的最大结合数为102，在pH 3.25时为82，在pH 4.35时为38，并详细分析了结合机制。研究了离子强度从0到1 mol L(-1)以及温度从20到70℃对结合的影响。结果表明，BRX与BSA的相互作用符合朗缪尔吸附等温模型，并测定了结合常数。根据结合数与碱性氨基酸残基数之间的相关性，阐明了离子对吸引诱导包括氢键、范德华力和疏水键在内的非共价键结合的结合模型。通过圆二色性证实，BRX与BSA的结合导致了BSA构象的改变。利用pH 2.03时的这种相互作用，建立了一种灵敏的吸光度比差光谱法并应用于蛋白质测定，蛋白质的检测限仅为6 μg L(-1)。对两个样品进行了测定，结果与经典考马斯亮蓝比色法所得结果一致。
