Buffer selection for HP treatment of biomaterials and its consequences for enzyme inactivation studies

Biochemical systems are best studied under buffered conditions and it is, therefore, necessary to have good knowledge on the pressure-induced changes in buffer pH. For experiments conducted at a set temperature, it is possible to use a relatively stable pressure buffer. However, when experiments have to be performed in a relatively large window of both pressure and temperature, it is not possible to find a buffer that is both pressure and temperature independent. An example of such an experiment is the measurement of pressure and temperature stability of enzymes. Here, we present the pressure dependence of pH of several buffers. We show that the pH of MES buffer pH 6.0 in a P-T plane from 0.1 to 1000 MPa and 10-90 °C has a pH range from 5.5 to 6.5. The effect of pH changes on enzyme inactivation is illustrated by an inactivation experiment with the -glycosidase from the hyperthermophile Pyrococcus furiosus. Within the measured range, a decrease in pH of 0.5 units causes the enzyme inactivation constant to increase by an additional factor 2-3