Blouin C, Guillemette J G, Wallace C J
Department of Biochemistry and Molecular Biology, Dalhousie University, Nova Scotia B3H 4H7, Canada.
Biophys J. 2001 Oct;81(4):2331-8. doi: 10.1016/S0006-3495(01)75879-1.
This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.
本通讯介绍了一种从复杂滴定曲线确定微观电离pK值的简单方法。我们用这种方法研究了线粒体细胞色素c的碱性转变（pH依赖的配体交换）。滴定曲线的线性化使得两到三个极限微观电离得以分辨。通过将这些数据与配体交换平衡的温度依赖性研究相结合，我们发现有证据表明碱性转变包括两个化学性质不同的过程：替代配体的去质子化和铁-甲硫氨酸配位键的断裂。我们还注意到，在马和未甲基化的酿酒酵母同工型1细胞色素c中，Lys(72)的ε-氨基可允许的去质子化使得在较低pH下形成碱性异构体，但其稳定性较低，这导致滴定曲线出现滞后现象。因此，不同细胞色素c滴定曲线的线性化有助于深入了解对构象稳定性的微观贡献。
