Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease.
Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease.
复制标题
低浓度变性剂对葡萄球菌核酸酶可逆变性的影响。
DOI：
10.1016/0003-9861(89)90200-2
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发表时间：
1989
影响因子：
3.9
通讯作者：
Gerring,SL
中科院分区：
文献类型：
--
作者：
Shortle,D;Meeker,AK;Gerring,SL
三个非常不稳定的突变形式的葡萄球菌核酸酶被用来定量的可逆变性（K app）的表观平衡常数的变化作为变性剂浓度的函数的各种不同的变性溶质。该平衡常数的值在变性剂（K app，0）的情况下，通过与甘油和钙离子的组合，后者结合在活性位点的天然构象的突变体蛋白质的复性确定。由于K app，0落在0.1和1之间的容易测量的范围内，因此可以在非常低的变性剂浓度下准确测量K app的变化，从而测量自由能（ΔG app）的变化。对于盐酸胍（GuHCl），发现变性的表观自由能相对于变性剂浓度（d（Δ G app）dC GuHCl或m GuHCl）的变化率在变性剂浓度为零时显著恒定，尽管该值对于三种蛋白质中的每一种都是不同的。与盐酸胍不同，尿素在低浓度下表现出d Δ G app dC尿素值略微降低。一些硫氰酸盐、高氯酸盐和碘化物的结果证实，霍夫迈斯特系列适用于浓度低于0.1 m的情况;也就是说，作为变性剂SCN−的有效性。ClO 4−> I−和Li+、NH 4+> Na+、K+。然而，分析的所有离液盐在浓度低于0.2 m时均表现出d（Δ G app）dC盐值显着增加。对于ΔG app和盐浓度之间的线性关系的这些大偏差的一个可能的解释是，离液序列高的阴离子的弱结合或吸附发生在变性状态的疏水区域中的饱和数量的位点处。
Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (K app) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (K app, 0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because K app, 0 fell in the easily measurable range between 0.1 and 1, the change in K app, and thus the change in free energy (ΔG app), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration (d (Δ G app) dC GuHCl or m GuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of d Δ G app dC urea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 m; that is, with regard to efficacy as a denaturant SCN−. ClO 4−> I− and Li+, NH 4+> Na+, K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d (Δ G app) dC salt at concentrations below 0.2 m. One possible explanation for these large deviations from a linear relationship between ΔG app and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.