Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.
Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.
复制标题
核糖核酸酶 S 中 3H 标记的 S 肽的浓度依赖性氢交换动力学。
DOI：
10.1016/0022-2836(76)90101-7
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发表时间：
1976
影响因子：
5.6
通讯作者：
R. L. Baldwin
中科院分区：
文献类型：
--
作者：
A. Schreier;R. L. Baldwin
核糖核酸酶S中S-肽的氢交换动力学可以通过首先在S-蛋白不存在的情况下氚化S-肽，然后使其与S-蛋白快速重组来测量。然后，可以研究核糖核酸酶S的这一特定片段的交换反应。结合的S-肽的交换动力学是复杂的，表明不同的质子以明显不同的速率交换。末端交换反应，涉及至少五个高度保护的质子，已被研究作为pH值的函数。在低浓度的核糖核酸酶S的交换动力学成为浓度依赖性的，由于S-肽的解离。虽然游离S-肽的分数总是非常小，但其交换速率比结合S-肽快几个数量级，并且交换动力学的浓度依赖性是容易测量的。它提供了一个高灵敏度的方法来确定小的解离常数（KD）。本文报告的KD值范围为10− 6 mat pH 2.7，0 °C至2 × 10− 10 mat pH 7.0，0 °C。我们在pH 7.0，0 °C下的Kd值证实了Hearnet等人的数据和外推到0 °C的结果。在高浓度的核糖核酸酶S下，末端交换反应与浓度无关。这可能是由于结合的S-肽的局部解折叠反应。高于pH 4的强pH依赖性ofKD密切类似于该局部展开反应的表观平衡常数。后者可能是在解离过程中的一个步骤，我们提出了这样一个模型核糖核酸酶S dissociation.Measurement浓度依赖性交换动力学应提供一个有用的方法，确定在其他系统中的小解离常数：例如，在蛋白质-核酸相互作用的研究。
The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (KD). Values ofKDranging from 10−6mat pH 2.7, 0 °C, to 2 × 10−10mat pH 7.0, 0 °C, are reported here. Our value forKDat pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearnet al.(1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence ofKDclosely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.