Gupta R, Yadav S, Ahmad F
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India.
Biochemistry. 1996 Sep 10;35(36):11925-30. doi: 10.1021/bi961079g.
We have studied the denaturation of metmyoglobin at pH 6.0 and 25 degrees C by urea and guanidine hydrochloride (GdnHCl) which are known to unfold the protein to the same extent. It has been observed that estimates of protein stability (delta G0N-U) from urea-induced and GdnHCl-induced denaturations do not agree with one another; the linear extrapolation method gave delta G0N-U values of 7.59 +/- 0.33 and 5.35 +/- 0.10 kcal mol-1 for urea and GdnHCl denaturations, respectively. Measurements of the effect of the addition of KCl in the concentration range 0.1-1.0 M to urea denaturation have suggested that this disagreement is not due to the nonionic and ionic characters of urea and GdnHCl, respectively. The functional dependence of the free energy change of unfolding (delta GN-U) on [denaturant], the molar concentration of the denaturant, has been investigated for understanding the cause(s) of the disagreement between the two estimates of delta G0N-U of metmyoglobin. For this purpose, we have studied the GdnHCl-induced denaturation of the protein in the presence of different urea concentrations at pH 6.0 and 25 degrees C and vice versa. These measurements yield delta GN-U values in the full concentration range [Ahmad et al. (1994) J. Biochem. 115, 322-327], and these results provide strong evidence that the delta GN-U dependence on [urea] is linear (linear free energy model of denaturation) and the relation between delta GN-U and [GdnHCl] is curved (binding model of denaturation). It has been observed that the extrapolated value of delta GN-U in urea using the linear free energy model becomes identical to the extrapolated value of delta GN-U in GdnHCl using the binding model.
我们研究了在pH 6.0和25℃条件下，已知能使蛋白质展开程度相同的尿素和盐酸胍（GdnHCl）对高铁肌红蛋白的变性作用。据观察，由尿素诱导和GdnHCl诱导变性得到的蛋白质稳定性估计值（ΔG0N-U）彼此不一致；线性外推法得出尿素变性和GdnHCl变性的ΔG0N-U值分别为7.59±0.33和5.35±0.10 kcal mol-1。对在0.1 - 1.0 M浓度范围内添加KCl对尿素变性影响的测量表明，这种不一致并非分别源于尿素和GdnHCl的非离子和离子特性。为了理解高铁肌红蛋白ΔG0N-U的两种估计值之间不一致的原因，研究了变性自由能变化（ΔGN-U）对变性剂摩尔浓度[变性剂]的函数依赖性。为此，我们研究了在pH 6.0和25℃条件下，不同尿素浓度存在时蛋白质的GdnHCl诱导变性，反之亦然。这些测量在整个浓度范围内得出了ΔGN-U值[Ahmad等人（1994年）《生物化学杂志》115卷，322 - 327页]，这些结果提供了有力证据，即ΔGN-U对[尿素]的依赖性是线性的（变性的线性自由能模型），而ΔGN-U与[GdnHCl]之间的关系是曲线的（变性的结合模型）。据观察，使用线性自由能模型在尿素中ΔGN-U的外推值与使用结合模型在GdnHCl中ΔGN-U的外推值相同。
