蛋白水解作用作为衡量细胞色素c与其衍生物之间自由能差异的一种方法。
Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives.
作者信息
Wang L, Kallenbach N R
机构信息
Department of Chemistry, New York University, New York 10003, USA.
出版信息
Protein Sci. 1998 Nov;7(11):2460-4. doi: 10.1002/pro.5560071124.
Abstract
Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.
摘要
在室温下，蛋白酶K对氧化型和还原型马心细胞色素c（Cyt c）以及Cyt c的叠氮化物复合物进行有限切割，会在中央环（序列中的36 - 60位）内产生单一切割位点。使用一种能对完整蛋白质的比例随时间进行光谱评估的测定方法，结合一个简单的蛋白水解动力学模型，环的波动打开可与相应蛋白质的自由能相关联。这使我们能够以蛋白水解为探针，定量估计Cyt c氧化形式与其他状态之间的自由能差。我们获得的结果表明，在25℃时，氧化型Cyt c比还原型稳定性低2.0千卡/摩尔，比Cyt c - 叠氮化物复合物稳定性高0.07千卡/摩尔。这些数值在量级上与氢交换和去折叠研究的结果一致，表明蛋白质的稳定性可直接与其结构动力学相关。
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