Protein stabilization by urea and guanidine hydrochloride
Protein stabilization by urea and guanidine hydrochloride
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DOI：
10.1021/bi020371n
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发表时间：
2002-11-12
期刊：
影响因子：
2.9
通讯作者：
Bhuyan, AK
中科院分区：
文献类型：
--
作者：
Bhuyan, AK
在pH为7的条件下，研究了CO与天然细胞铁色素c在非平衡状态下解离速率与尿素、盐酸胍、盐和温度的关系。在pH为7的0.1 M磷酸盐中制备的盐酸胍（GdnHCl）和尿素的存在下，铁细胞色素c的血红素铁与CO结合。当未折叠的蛋白质溶液被稀释101倍到不含CO的折叠缓冲液中时，蛋白质链完全重新折叠，使CO分子与血红素铁结合。随后，CO分子的缓慢热解离产生天然M80配体的血红素配位。因此，该反应监测co配体的天然铁细胞色素c到m80配体的天然蛋白质的热转化速率。该反应的速率k（diss）表现出与反应介质中存在非变性浓度的变性剂有关的特征。当还原介质中GdnHCl浓度从接近0增加到接近2.1 m时，还原速率降低了1.9-3倍。同样，当尿素浓度从0.1增加到接近5 m时，还原速率降低了1.8倍。在更高浓度的变性剂下，变性效应开始发挥作用，蛋白质不稳定，因此CO解离速率急剧增加。随着反应介质中变性剂浓度的增加，反应的活化能E-a增加：GdnHCl浓度增加0.05 ~ 2.1 M时，反应活化能E-a从24.1增加到28.3 kcal mol(-1)；尿素浓度增加0.1 ~ 26.9 M时，反应活化能E-a从25.2增加到26.9 kcal mol（-1）。与变性剂浓度的增加相对应的是活化熵S-diss/R的增加，其中R是反应的气体常数。这些CO解离反应的动力学和热力学参数对变性剂的依赖性表明，与GdnHCl和尿素的结合相互作用可以提高铁细胞色素c的结构和能量稳定性，达到添加剂亚变性浓度的极限。NaCl和Na2SO4通过盐化作用稳定蛋白质，也降低了CO与CO结合的天然铁细胞色素c解离的激活熵，从而支持了低浓度GdnHCl和尿素稳定蛋白质的观点。这些结果对自由能变性关系和蛋白质折叠曲线的理解和解释具有直接意义。
The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by similar to1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to similar to2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.1 to similar to5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E-a, increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S-diss/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na2SO4, which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.