The role of pH change caused by the addition of water-miscible organic solvents in the destabilization of an enzyme

Abstract

The possibility that a change in the pH of a buffer caused by addition of organic solvents can play a role in the destabilization of an enzyme in aqueous mixtures of polar solvents was studied. The magnitude of change in pH was dependent on the concentration of the organic solvent, the nature of the buffer ions, and the concentration and initial pH of the buffer. The decrease in pH of sodium borate buffer caused by the addition of polyhydroxy compounds was used to counteract the pH increase due to organic solvents. The stability and activity of trypsin, and β-galactosidases from Aspergillus oryzae and Escherichia coli in aqqueous NN-dimethyl formamide (DMF) with or without mannitol were compared with those at analogous pH values in aqueous solution. The stability and activity of β-galactosidase from A. oryzae in aqueous mixtures containing low concentrations of DMF paralleled those in buffers without DMF at comparable pH, whereas at higher concentrations of DMF the activity and stability were much lower. No such correlation was observed in the case of trypsin or β-galactosidase from E. coli. Therefore, at a lower concentration of organic solvents, the change in pH of the buffer plays a major role in affecting the stability and activity of A. oryzae β-galactosidase. As the concentration of the solvent increases, its direct interaction with the protein becomes the dominating factor in the denaturation of the enzyme.