Galisteo M L, Mateo P L, Sanchez-Ruiz J M
Departamento de Quimica Fisica (Facultad de Ciencias) e Instituto de Biotecnologia, Universidad de Granada, Spain.
Biochemistry. 1991 Feb 26;30(8):2061-6. doi: 10.1021/bi00222a009.
Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.
在pH 7.0条件下，酵母磷酸甘油酸激酶不可逆热变性的差示扫描量热转变强烈依赖于扫描速率，这表明变性至少部分受动力学控制。为了验证这种可能性，我们对该酶的热失活进行了动力学研究。在量热转变的温度范围内，失活动力学相对较快，并且可以用现象学方程dC/dt = -αC²/(β + C)来描述，其中C是给定时间t时活性酶的浓度，α和β是取决于温度的速率系数。这个方程，连同α和β的值（在50 - 59摄氏度的温度范围内），使我们能够计算出与量热实验相对应的不可逆变性蛋白质分数与温度的关系曲线。我们发现：(a) 不可逆变性发生在蛋白质处于转变区域的时间段内；(b) 量热转变最大值的温度（Tm）与一半蛋白质发生不可逆变性的温度（Th）之间存在极好的相关性。这些结果表明，磷酸甘油酸激酶变性的差示扫描量热转变被限速不可逆过程严重扭曲。最后，对平衡热力学在不可逆蛋白质变性分析中的应用作了一些评论。
