Carvalho Ana Sofia L, Melo Eduardo Pinho e, Ferreira Bruno Sommer, Neves-Petersen Maria Teresa, Petersen Steffen B, Aires-Barros Maria Raquel
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av Rovisco Pais, Lisboa 1049-001, Portugal.
Arch Biochem Biophys. 2003 Jul 15;415(2):257-67. doi: 10.1016/s0003-9861(03)00275-3.
Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.
在pH值为4、7和10的条件下，通过稳态荧光、圆二色性和差示扫描量热法研究了辣根过氧化物酶A1的热稳定性。蛋白质内在探针、色氨酸荧光、二级结构和血红素基团环境的变化并不一致。在所有pH值下，从可见圆二色性数据测得的Tm值均高于从色氨酸荧光和远紫外圆二色性数据测得的Tm值，这表明在热变性过程中，血红素腔是最后一个发生显著构象变化的结构区域。然而，在pH值为4时，血红素基团的排出会导致不可逆的去折叠行为，而在pH值为7和10时，仍可观察到再折叠现象。这可能与血红素口袋的滴定状态有关。HRPA1的热转变在三个pH值下均表现出扫描速率依赖性，表明变性过程受动力学控制。根据经典方案N<-->U-->D对变性过程进行了解释，并将其拟合到远紫外圆二色性椭圆率。实验和理论的Tm值以及不可逆百分比之间取得了良好的一致性。然而，正如多个Tm值所揭示的那样，N和U之间的平衡可能比简单的两态过程更为复杂。
