SOLUBILITY AND SECONDARY STRUCTURE OF BOVINE PANCREATIC -CHYMOTRYPSIN IN WATER{ACETONITRILE MIXTURES

Solubility of bovine pancreatic -chymotrypsin (CT) in water-acetonitrile mixtures was measured using UV-spectroscopy at 298 K. Water activity in mixture was varied from 0.34 to 1.0. The protein secondary structure was controlled in the same range of compositions by FTIR spectroscopy. It was found that the CT solubility depend on water activity in threshold manner. At water activity range below 0.7 the enzyme is completely insoluble and its secondary structure is close to that of air-dried protein. In the activity range from 0.75 to 0.92 solubility rises sharply. Secondary structure of CT in this range both in solution and precipitate was characterized as denatured one with predominate intermolecular -structure due to extensive aggregation. Above aw = 0:92 enzyme remains completely soluble at the protein concentration employed and its secondary structure is close to that in pure water. Comparing with the data on water state in acetonitrile at the same activity range we could explain the changes in CT solubility and structure as a result of the following factors: progressive disruption of spatial hydrogen bond network of water molecules and lowering of conformational motility of the protein molecule as water activity diminish.