Thermodynamics of the Hydrophobicity in Crystallization of Insulin

Abstract

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, C ac = 0%, 5%, 10%, 15%, and 20%, and find that, as a rule, the solubility of insulin increases as temperature increases. The enthalpy of crystallization, Δ H cryst o , undergoes a stepwise shift from ∼−20 kJ mol −1 at C ac = 0%, 5%, and 10% to ∼−55 kJ mol −1 at C ac = 15% and 20%. The entropy change upon crystallization Δ S cryst o > 0 is ∼35 J mol −1 K −1 for the first three acetone concentrations, and drops to ∼−110 J mol −1 K −1 at C ac = 15% and 20%. Δ G cryst o indicates release of solvent, mostly water, molecules structured around the hydrophobic patches on the insulin molecules’ surface in the solution. As C ac increases to 15% and above, unstructured acetone molecules apparently displace the waters and their contribution to Δ S cryst o > 0 is minimal. This shifts Δ S cryst o > 0 to a negative value close to the value expected for tying up of one insulin molecule from the solution. The accompanying increase in Δ H cryst o 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.