Santra Manas Kumar, Banerjee Abhijit, Krishnakumar Shyam Sundar, Rahaman Obaidur, Panda Dulal
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai, India.
Eur J Biochem. 2004 May;271(9):1789-97. doi: 10.1111/j.1432-1033.2004.04096.x.
The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.
远紫外圆二色（CD）信号、内源性色氨酸荧光和胆红素吸光度的变化表明，盐酸胍（GdnHCl）诱导的多结构域蛋白人血清白蛋白（HSA）的去折叠遵循两态过程。然而，使用对环境敏感的尼罗红荧光，发现HSA的去折叠和折叠途径遵循三态过程，并且在0.25 - 1.5 m GdnHCl范围内检测到一个中间体。中间体状态的荧光强度比天然状态高45%。发现尼罗红荧光的增加是由于与HSA结合的尼罗红的量子产率增加。低浓度的GdnHCl既不改变尼罗红与HSA的结合亲和力，也不诱导HSA聚集。此外，在第一次去折叠转变（<1.5 m GdnHCl）期间，HSA的二级结构未受到扰动；然而，在第二次转变期间二级结构完全丧失。这些数据共同表明，三级结构的半最大丧失发生在比二级结构丧失更低的GdnHCl浓度下。使用多种光谱技术对HSA复性过程进行的进一步动力学研究表明，复性分两个阶段进行，一个快速阶段之后是一个缓慢阶段。发现在复性的快速阶段形成了一种具有类似天然二级结构但只有部分三级结构的中间体。然后，中间体缓慢折叠成天然状态。对复性数据的分析表明，HSA的折叠可以用框架模型得到最好的解释。
