Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up t o 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as wel l as aggregation state. Tertiary structure was monitored in situ with secon d derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and p otential secondary structural changes were characterized by second derivati ve FTIR spectroscopy. Changes in aggregation state were assessed using dyna mic light scattering. Additionally, protein concentration balances were car ried out to detect any loss of protein as a function of pressure. For the c onditions tested, neither protein shows measurable changes in tertiary or s econdary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein inte raction with the vial surface and appear to be rate Limited by the equilibr ium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.