pH determination by pyranine: Medium-related artifacts and their correction

Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) is a water-soluble, membrane-impermeable fluorophore having fluorescent excitation and emission spectra that are highly dependent on medium pH. This combination makes it one of the most commonly used pH-sensitive fluorescent probes to monitor pH and pH changes in biochemical and biophysical research. The pK_a of this probe is reported to be approximately 7.3, but several studies (including the current one) have shown that this value varies with medium composition. If this is not taken into account, pH determinations based on pyranine may be misleading. We found that in the presence of salts, pK_a is shifted downward to lower values; therefore, the calculated pH is shifted upward relative to the actual pH as determined by a pH meter. This shift is a consequence of both the type and the concentration of anions and cations that form the salt. Divalent cations cause a larger upward shift in the calculated pH than do monovalent cations. Of all the salts tested, ammonium sulfate has the least effect, and calcium perchlorate has the largest effect, on the pH value calculated by pyranine. Salts are not the only species that affect the pK _a of pyranine. The presence of the polymer polyethylene glycol (PEG) induces an effect opposite to that of salt (i.e., an upward pK_a shift), which is expressed as pH being more acidic than that measured by a pH meter. Another nonelectrolyte, dextrose, has no such effect. The effect of both cations and anions can be explained based on their order in the Hofmeister series, whereas the effect of PEG is explained by its high water-binding capacity. Both the ions and PEG change the structure of water and its interaction with pyranine, thereby changing pyranine's apparent pK_a.