INFLUENCE OF TRANSITION RATES AND SCAN RATE ON KINETIC SIMULATIONS OF DIFFERENTIAL SCANNING CALORIMETRY PROFILES OF REVERSIBLE AND IRREVERSIBLE PROTEIN DENATURATION
INFLUENCE OF TRANSITION RATES AND SCAN RATE ON KINETIC SIMULATIONS OF DIFFERENTIAL SCANNING CALORIMETRY PROFILES OF REVERSIBLE AND IRREVERSIBLE PROTEIN DENATURATION
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DOI：
10.1021/bi00165a023
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发表时间：
1992-12-22
期刊：
影响因子：
2.9
通讯作者：
KRUUV, J
中科院分区：
文献类型：
--
作者：
LEPOCK, JR;RITCHIE, KP;KRUUV, J
利用差示扫描量热法（DSC）可以直接获得表征蛋白质折叠的热力学参数。它们仅对平衡时的可逆展开有意义，这适用于小球状蛋白;然而，大多数大的多结构域或多亚基蛋白的展开或变性是部分或完全不可逆的。描述部分不可逆变性的最简单的动力学模型需要三种状态：[图形]我们获得了N，U和D的数值解，作为该模型的温度函数，并根据约化变量v/k（i）和k1/k3推导出过量比热（C（p））的曲线，其中v是扫描速率。对于很大或很小的k1/k3值，三态模型分别简化为两态可逆或不可逆模型。表观转变温度（T（app））总是被不可逆步骤（U -> D）降低。对于k3的所有值，T（app）是独立的v/k1在足够慢的扫描速率，即使当变性是高度不可逆的，但增加相同的所有模型在快速扫描速率，在这种情况下，过量的比热分布是由解折叠的速率。只有当k_1大于k_3的2000 ~ 50000倍时，才能得到可逆阶跃的Δ H和Δ TAS的精确值。原则上，比率k1/k3的近似值可以从作为温度的函数的未折叠部分与不可逆变性部分的图中获得;然而，不可逆变性部分难以单独通过DSC精确测量。一个指南，以确定是否可逆的分析是适当的分析DSC扫描明显不可逆变性的基础上，这些图描述。虽然在大于95%解折叠时可以容忍高水平的不可逆性，但在较低水平的解折叠时，对于使用假定可逆性的模型精确测定DELTAH和DELTAS以及复杂曲线的去卷积，仅可以发生非常低水平的不可逆性（在75%解折叠时为1-14%，在50%解折叠时小于2%）。在不可逆性较高的条件下，动力学分析更合适，信息量也更大。
The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states:[GRAPHICS]We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (C(p)) in terms of the reduced variables v/k(i) and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (T(app)) is always reduced by the irreversible step (U --> D). For all values of k3, T(app) is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of DELTAH and DELTAS can be obtained for the reversible step only when k1 is more than 2000-50 000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone. A guide to determining whether a reversible analysis is appropriate for analyzing DSC scans for apparently irreversible denaturation is described on the basis of these plots. While a high level of irreversibility can be tolerated at greater than 95% unfolding, at lower levels of unfolding only very low levels of irreversibility (1-14% at 75% unfolding and less than 2% at 50% unfolding) can occur for accurate determination of DELTAH and DELTAS and deconvolution of complex profiles using models that assume reversibility. Under conditions of higher irreversibility, a kinetic analysis is both more appropriate and more informative.