Schubert Frank, Zettl Heiko, Häfner Wolfgang, Krauss Gerhard, Krausch Georg
Physikalische Chemie II, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany.
Biochemistry. 2003 Sep 2;42(34):10288-94. doi: 10.1021/bi034033d.
We report a kinetic and thermodynamic analysis of interactions between ssDNA and replication protein A (RPA) using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at variable temperature. The two methods yield different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA-RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.6 and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.4 and -66.5 kJ/mol. It is concluded that the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. The entropic contribution to the free energy at 25 degrees C is -1.8 kJ/mol for SPR and -11.8 kJ/mol for FCS. In SPR, the reaction is restricted to two dimensions because of immobilization of the DNA molecules to the sensor surface. In contrast, FCS is able to follow complex formation without spatial restrictions. In consequence, the reaction entropy determined from SPR experiments is lower than for FCS experiments.
我们报告了在可变温度下使用表面等离子体共振（SPR）和荧光相关光谱（FCS）对单链DNA（ssDNA）与复制蛋白A（RPA）之间相互作用进行的动力学和热力学分析。这两种方法得出的吉布斯自由能值不同，但对于ssDNA-RPA复合物形成的反应焓值几乎相同。通过SPR和FCS测定的吉布斯自由能分别为-62.6和-54.7 kJ/mol。反应焓值分别为-64.4和-66.5 kJ/mol。得出的结论是，两种方法测量的吉布斯自由能差异是由于反应熵不同。在25摄氏度时，熵对自由能的贡献对于SPR为-1.8 kJ/mol，对于FCS为-11.8 kJ/mol。在SPR中，由于DNA分子固定在传感器表面，反应被限制在二维空间。相比之下，FCS能够在没有空间限制的情况下跟踪复合物的形成。因此，从SPR实验确定的反应熵低于FCS实验的反应熵。
