Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2.
Biophys Chem. 2010 May;148(1-3):144-7. doi: 10.1016/j.bpc.2010.03.002. Epub 2010 Mar 12.
Protein denaturation events are generally associated with a change in the state of ionization of abnormally titrating groups and, therefore, are coupled with changes in buffer ionization/neutralization equilibria. Consequently, buffer ionization should influence the measured change in volume accompanying protein denaturation. Changes in volume accompanying protein denaturation reflect the differential packing and hydration of polypeptide chains in their native and denatured conformations while also describing the pressure stability of proteins. A characteristic feature of conformational transitions of globular proteins is a near zero change in volume that is comparable in magnitude with the volume of ionization of biologically relevant buffers. Thus, the impact of buffer ionization on the volume of protein denaturation could be very significant with the potential to affect not only its magnitude but also its sign. To investigate this point quantitatively, we performed pressure perturbation calorimetric (PPC) studies of lysozyme and ribonuclease A at pH 3.0 in four buffers differing in their ionization volumes. Our results identify buffer ionization as an important determinant of protein transition volume that needs to be carefully taken into account. We emphasize that the importance of our results is not limited to PPC measurements but is more general and applies to all volumetric investigations, in particular, extending to the derivation of the pressure-temperature phase diagram of protein stability.
蛋白质变性事件通常与异常滴定基团的电离状态变化有关，因此与缓冲液电离/中和平衡的变化相关。因此，缓冲液电离应该会影响伴随蛋白质变性的体积测量变化。伴随蛋白质变性的体积变化反映了多肽链在其天然和变性构象中的差异包装和水合，同时也描述了蛋白质的压力稳定性。球状蛋白质构象转变的一个特征是体积几乎不变，与生物相关缓冲液的电离体积相当。因此，缓冲液电离对蛋白质变性体积的影响可能非常显著，不仅可能影响其大小，还可能影响其符号。为了定量研究这一点，我们在 pH 3.0 下的四个缓冲液中进行了溶菌酶和核糖核酸酶 A 的压力微扰量热法（PPC）研究，这些缓冲液在其电离体积上有所不同。我们的结果表明，缓冲液电离是蛋白质转变体积的一个重要决定因素，需要仔细考虑。我们强调，我们的结果的重要性不仅限于 PPC 测量，而是更普遍，适用于所有体积研究，特别是扩展到蛋白质稳定性的压温相图的推导。
