Bergeron Lisa, Filobelo Luis F, Galkin Oleg, Vekilov Peter G
Department of Chemical Engineering, University of Houston, Houston, Texas 77204-4004, USA.
Biophys J. 2003 Dec;85(6):3935-42. doi: 10.1016/S0006-3495(03)74807-3.
For insight into the solvent structure around protein molecules and its role in phase transformations, we investigate the thermodynamics of crystallization of the rhombohedral form of porcine insulin crystals. We determine the temperature dependence of the solubility at varying concentration of the co-solvent acetone, Cac=0%, 5%, 10%, 15%, and 20%, and find that, as a rule, the solubility of insulin increases as temperature increases. The enthalpy of crystallization, undergoes a stepwise shift from approximately -20 kJ mol(-1) at Cac=0%, 5%, and 10% to approximately -55 kJ mol(-1) at Cac=15% and 20%. The entropy change upon crystallization is approximately 35 J mol(-1) K(-1) for the first three acetone concentrations, and drops to approximately -110 J mol(-1) K(-1) at Cac=15% and 20%. DeltaS degrees cryst>0 indicates release of solvent, mostly water, molecules structured around the hydrophobic patches on the insulin molecules' surface in the solution. As Cac increases to 15% and above, unstructured acetone molecules apparently displace the waters and their contribution to DeltaS degrees cryst is minimal. This shifts DeltaS degrees cryst to a negative value close to the value expected for tying up of one insulin molecule from the solution. The accompanying increase in DeltaH degrees cryst suggests that the water structured around the hydrophobic surface moieties has a minimal enthalpy effect, likely due to the small size of these moieties. These findings provide values of the parameters needed to better control insulin crystallization, elucidate the role of organic additives in the crystallization of proteins, and help us to understand the thermodynamics of the hydrophobicity of protein molecules and other large molecules.
为深入了解蛋白质分子周围的溶剂结构及其在相变中的作用，我们研究了猪胰岛素晶体菱形晶型的结晶热力学。我们测定了在共溶剂丙酮浓度Cac = 0%、5%、10%、15%和20%变化时溶解度随温度的变化，发现通常胰岛素的溶解度随温度升高而增加。结晶焓在Cac = 0%、5%和10%时约为 -20 kJ·mol⁻¹，逐步转变为在Cac = 15%和20%时约为 -55 kJ·mol⁻¹。对于前三种丙酮浓度，结晶时的熵变约为35 J·mol⁻¹·K⁻¹，在Cac = 15%和20%时降至约 -110 J·mol⁻¹·K⁻¹。ΔS°cryst > 0表明溶剂（主要是水）分子从溶液中胰岛素分子表面疏水区域周围的结构化状态释放出来。随着Cac增加到15%及以上，无结构的丙酮分子显然取代了水分子，它们对ΔS°cryst的贡献最小。这使得ΔS°cryst变为负值，接近从溶液中结合一个胰岛素分子所预期的值。伴随的ΔH°cryst增加表明，疏水表面部分周围的水具有最小的焓效应，可能是由于这些部分的尺寸较小。这些发现提供了更好控制胰岛素结晶所需的参数值，阐明了有机添加剂在蛋白质结晶中的作用，并帮助我们理解蛋白质分子和其他大分子疏水性的热力学。
