Yang H P, Zhong H N, Li S, Zhou H M
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, P. R. China.
Biochem Mol Biol Int. 1997 Feb;41(2):257-67. doi: 10.1080/15216549700201261.
The conformational changes of creatine kinase during alkaline unfolding and salt-induced folding at high pH have been followed by fluorescence emission and circular dichroism spectra. The results obtained show that at low ionic strength, with increasing pH value, creatine kinase denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 12.7. With the increase of pH from 9.0 to 12.7, the fluorescence emission maximum red shifted from 337 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far-UV CD spectra show that even at pH 12.7, the apparently fully denatured enzyme retains a great part of ordered secondary structure. At pH 12.7 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded state induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. The above results suggest that the salt-induced folded state at high pH may be the folded intermediate which exists in the general protein folding, and the larger residual ordered secondary structure might become folded being point on the salt-induced folding.
通过荧光发射光谱和圆二色光谱跟踪了肌酸激酶在碱性展开和高pH值下盐诱导折叠过程中的构象变化。所得结果表明，在低离子强度下，随着pH值升高，肌酸激酶逐渐变性，在pH 12.7附近达到最终的未折叠构象。随着pH从9.0增加到12.7，荧光发射最大值从337 nm红移至355 nm，表明埋藏的色氨酸残基完全暴露于溶剂中。远紫外圆二色光谱表明，即使在pH 12.7时，明显完全变性的酶仍保留很大一部分有序二级结构。在pH 12.7时加入盐，变性酶相对未折叠的状态通过疏水塌缩转变为紧密的构象状态。盐结合的ANS强烈诱导折叠状态，表明存在增加的疏水表面。上述结果表明，高pH值下盐诱导的折叠状态可能是一般蛋白质折叠中存在的折叠中间体，较大的残余有序二级结构可能成为盐诱导折叠的关键点。
