Utilization of dual-wavelength pH-sensing dyes in the characterization of pH buffers under hydrostatic pressure

Biologically-compatible pH-buffer solutions are routinely used in biochemical and biophysical studies at ambient pressure. When using buffers under high-pressure however, the existence of a thermodynamic volume change ($\Delta $V) for the buffer's acid-dissociation reaction leads to a pressure-dependent equilibrium constant (pK$_{\mathrm{a}})$. The result is a pressure-induced shift in pH. Here we use pH-sensing, dual-wavelength probe dyes, SNARF-1 and SNARF-5F, to characterize the pressure-response of three biologically-relevant buffers: MOPS, HEPES, and TRIS. Emission spectra from the dyes, excited at 337-nm wavelength, are used to track the pressure-induced change in pH of the buffer systems, up to 600 atm (61 MPa). From this the $\Delta $V of the buffers' acid-dissociation reaction was determined and found to be in the 0 to $+$10 ml/mol range. Results presented are important when accounting for pressure-induced pH changes during high-pressure biophysical studies because a large range of biochemical reactions involve H$^{\mathrm{+}}$ and have a $\Delta $V similar in range to the these buffers.