Guo Shu-Yuan, Guo Zhi, Chen Bao-Yu, Guo Qin, Ni Shao-Wei, Wang Xi-Cheng
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084, China.
Biochemistry (Mosc). 2003 Nov;68(11):1267-71. doi: 10.1023/b:biry.0000009143.92546.33.
Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.
采用尿素滴定法研究了仿刺参精氨酸激酶（AK）的失活和去折叠平衡。荧光光谱和圆二色光谱数据均表明，在1.0至2.0 M尿素存在下，AK存在去折叠中间体。尺寸排阻色谱结果进一步支持了这一点。光谱数据表明，这种去折叠中间体与AK天然形式在二级结构、三级结构以及四级结构等方面具有许多结构特征。此外，根据残余活性曲线，这种去折叠中间体形式虽然活性低于天然AK，但仍保留其催化功能。综上所述，我们的研究结果直接证明，在用尿素滴定过程中，AK去折叠平衡状态存在具有部分活性的中间体。
