Myer Y P
J Biol Chem. 1984 May 25;259(10):6127-33.
The refolding of urea-denatured horse heart ferricytochrome c in the presence of imidazole, 0.5 M, pH 7.0, has been examined using stopped-flow and equilibrium measurements at 407.5 nm. Thermodynamically, imidazole-cytochrome c folds and unfolds via a single transition with [urea]1/2 of 5.9 M. Kinetically, the refolding is a triphasic process: (i) a slow, urea-independent phase, time constant of 22 +/- 6 s, and an amplitude of 10-13%; (ii) an intermediate reaction, with a slightly positive urea-dependent rate constant, average time constant of 150 ms; and (iii) a fast phase with negative urea dependence of the rate constant from 4-6 M urea and positive dependence above the 6 M concentration, with the largest time constant, 25 +/- 6 ms, at 5.8 M urea, the midpoint of the transition. The amplitudes of the intermediate and the fast phases exhibit inverse dependence on the final urea concentrations, favoring the intermediate form at higher concentrations, while maintaining an almost constant sum of the two amplitudes throughout the range. The temperature dependence of the three apparent rate constants for the refolding from denatured base-line to midpoint of the transition, 9 to 6.03 M urea, yields linear Arrhenius plots with activation energies of 14, 19, and 23 +/- 3 kcal/mol for the slow, intermediate, and rapid reactions, respectively. These findings show that the slow reaction, time constant in decaseconds , does not require, directly or indirectly, the coordination of Met-80-S to heme iron. The formation of this linkage during the folding of the urea-denatured protein in the absence of extrinsic ligand, however, does alter the course of the refolding process. From a comparison of the proposed mechanisms and of the kinetic parameters for the folding of urea-denatured and of guanidine hydrochloride-denatured ferricytochrome c, it has been suggested that the two systems are distinct in detail, although both systems exhibit the slow, decasecond process.
在0.5M、pH7.0的咪唑存在下，使用停流法和在407.5nm处的平衡测量研究了尿素变性的马心铁细胞色素c的复性。从热力学角度看，咪唑 - 细胞色素c通过单一转变进行折叠和展开，[尿素]1/2为5.9M。从动力学角度看，复性是一个三相过程：（i）一个缓慢的、与尿素无关的相，时间常数为22±6秒，幅度为10 - 13%；（ii）一个中间反应，具有略微正的尿素依赖性速率常数，平均时间常数为150毫秒；（iii）一个快速相，速率常数在4 - 6M尿素时对尿素呈负依赖性，在6M浓度以上呈正依赖性，在5.8M尿素（转变中点）时时间常数最大，为25±6毫秒。中间相和快速相的幅度对最终尿素浓度呈反依赖性，在较高浓度时有利于中间形式，同时在整个范围内保持两个幅度的总和几乎恒定。从变性基线到转变中点（9至6.03M尿素）复性的三个表观速率常数的温度依赖性，分别给出了慢、中、快反应的线性阿伦尼乌斯图，活化能分别为14、19和23±3千卡/摩尔。这些发现表明慢反应（时间常数在十秒量级）不直接或间接需要Met - 80 - S与血红素铁的配位。然而，在没有外在配体的情况下，尿素变性蛋白折叠过程中这种连接的形成确实改变了复性过程的进程。通过比较尿素变性和盐酸胍变性的铁细胞色素c折叠的拟议机制和动力学参数，表明尽管两个系统都表现出缓慢的、十秒量级的过程，但在细节上是不同的。
