DIFFERENTIAL SCANNING CALORIMETRIC STUDY OF THE THERMAL UNFOLDING OF BETA-LACTAMASE-I FROM BACILLUS-CEREUS
DIFFERENTIAL SCANNING CALORIMETRIC STUDY OF THE THERMAL UNFOLDING OF BETA-LACTAMASE-I FROM BACILLUS-CEREUS
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DOI：
10.1021/bi00143a034
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发表时间：
1992-07-21
期刊：
影响因子：
2.9
通讯作者：
LAYNEZ, J
中科院分区：
文献类型：
--
作者：
ARRIAGA, P;MENENDEZ, M;LAYNEZ, J
采用差示扫描量热法（DSC）和失活动力学技术研究了蜡状芽孢杆菌A类β-内酰胺酶I在pH 7.0时的不可逆热去折叠。DSC转变显示出一个单峰，变性焓为646 kJ.mol-1，并适度扫描速率依赖性，这表明该过程是部分动力学控制。在恒定温度下的失活动力学表明，酶的不可逆变性发生的两个指数项的总和，其幅度是强烈的温度依赖性的过渡范围内，因此，在此区间内的最低温度下，不可逆失活将主要通过缓慢相进行。使用先前确定的动力学参数，通过动力学方程与温度的数值积分，计算对于给定扫描速率作为温度的函数的不可逆变性酶（D）的分数。这种D型只有在远高于量热跃迁最大值的温度下，才是最常见的未折叠态。动力学和DSC实验结果的结合使我们能够将最终D状态对过量焓变的贡献与酶的可逆变性形式（I（i），i = 1，.，n），得到的结论是量热迹线的扫描速率依赖性是两种不同动力学效应的结果，即，在可逆变性中间态形成过程中的不可逆步骤和缓慢弛豫过程。最后，使用在一个单一的扫描速率，以验证两个状态的动力学模型的结果的问题进行评论。
The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1, ..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was-the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.