UNFOLDING FREE-ENERGY CHANGES DETERMINED BY THE LINEAR EXTRAPOLATION METHOD .1. UNFOLDING OF PHENYLMETHANESULFONYL ALPHA-CHYMOTRYPSIN USING DIFFERENT DENATURANTS
UNFOLDING FREE-ENERGY CHANGES DETERMINED BY THE LINEAR EXTRAPOLATION METHOD .1. UNFOLDING OF PHENYLMETHANESULFONYL ALPHA-CHYMOTRYPSIN USING DIFFERENT DENATURANTS
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DOI：
10.1021/bi00421a014
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发表时间：
1988-10-18
期刊：
影响因子：
2.9
通讯作者：
BOLEN, DW
中科院分区：
文献类型：
--
作者：
SANTORO, MM;BOLEN, DW
对于苯甲磺酰基胰凝乳蛋白酶（PMS-Ct）的去折叠，评估了通过线性外推法测定的去折叠自由能变化Δ G ° N-U的特征和性质。在293 nm处的差光谱测量用于定义用氯化胍、尿素和1，3-二甲基脲引起的PMS-Ct解折叠。所有三种变性剂显示出给出相同的消光系数差（Δ. NU）之间的天然和未折叠形式的蛋白质在零浓度的变性剂的限制。△的独立性。NU on denaturant支持变性前和变性后基线线性延伸到过渡区，允许基于双态假设评估展开平衡常数。使用基于线性外推法的表达式，使用原始数据Δ的非线性最小二乘拟合来提供三种变性剂的Δ G ° N-U的估计值。- 是的相对于[变性剂]。三个Δ G ° N-U值在误差范围内是相同的，这表明自由能变化是蛋白质系统的性质并且与变性剂无关。建议使用线性外推法确定的Δ G ° N-U的误差显著大于文献中通常报道的误差。
Characteristics and properties of the unfolding free energy change, .DELTA.G.degree.N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (.DELTA..epsilon.NU) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of .DELTA..epsilon.NU on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of .DELTA.G.degree.N-U for the three denaturants using nonlinear least-squares fitting of the primary data, .DELTA..epsilon. versus [denaturant]. The three .DELTA.G.degree.N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in .DELTA.G.degree.N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.