Hernández-Arana A, Rojo-Domínguez A, Altamirano M M, Calcagno M L
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, México, D.F., Mexico.
Biochemistry. 1993 Apr 13;32(14):3644-8. doi: 10.1021/bi00065a017.
The thermal denaturation of Escherichia coli glucosamine-6-phosphate deaminase (G6PD) at neutral pH was studied by means of differential scanning calorimetry (DSC). In the concentration range 0.6-7.3 mg mL-1, the denaturation of this hexameric enzyme was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. However, the study of the effect of scanning rate on DSC curves indicated that the denaturation of G6PD is, most likely, a complex process which includes transitions in equilibrium as well as an irreversible step; in addition, it was found that application of the equilibrium formalism to the analysis of calorimetric data seems to be justified in this case, provided that scanning rates used are above 0.75 K min-1. The calorimetric and van't Hoff enthalpies for G6PD were 1260 +/- 118 and 160 +/- 27 kcal mol-1, respectively, indicating the presence of intermediates in the process. Accordingly, the DSC curves were adequately fitted to a model including six two-state sequential transitions. The observed protein-concentration dependence of the temperature at the maximum heat capacity, Tm, for each of the individual transitions suggests that G6PD dissociates to dimers in two consecutive steps. Using a model that includes dissociation explicitly, we calculated the thermodynamic parameters for each step. From this data, the enthalpy and free energy for the disruption of one dimer-dimer contact were roughly estimated, at pH 7.1 and 51 degrees C, as 57 and 2.1 kcal mol-1, respectively.
利用差示扫描量热法（DSC）研究了中性pH条件下大肠杆菌葡糖胺-6-磷酸脱氨酶（G6PD）的热变性。在0.6 - 7.3 mg mL-1的浓度范围内，通过对先前扫描样品进行再扫描时未出现任何吸热峰判断，这种六聚体酶的变性是完全不可逆的。然而，扫描速率对DSC曲线影响的研究表明，G6PD的变性很可能是一个复杂的过程，包括平衡转变以及一个不可逆步骤；此外，还发现，在这种情况下，只要使用的扫描速率高于0.75 K min-1，将平衡形式应用于量热数据的分析似乎是合理的。G6PD的量热焓和范特霍夫焓分别为1260±118和160±27 kcal mol-1，表明该过程中存在中间体。因此，DSC曲线能很好地拟合到一个包含六个两态连续转变的模型。观察到的每个单独转变的最大热容量温度Tm对蛋白质浓度的依赖性表明，G6PD以两个连续步骤解离为二聚体。使用一个明确包含解离的模型，我们计算了每个步骤的热力学参数。根据这些数据，在pH 7.1和51℃下，破坏一个二聚体-二聚体接触的焓和自由能分别大致估计为57和2.1 kcal mol-1。
