AJP - Cell Physiology, Vol 264, Issue 1 C27-C31, Copyright © 1993 by American Physiological Society
Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ
M. Vasseur, R. Frangne and F. Alvarado
Centre de Recherche sur l'Endocrinologie Moleculaire et le Developpement, Centre National de la Recherche Scientifique, Meudon, France.
The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)
in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)
2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane
buffer, pH 7.0, decreased as a function of Cl- concentration and/or
gluconate concentration, as expected. Contrary to expectation, however, the
fluorescence intensity progressively increased as the pH decreased,
independently of the presence of gluconate. Consequently, the modulation of
SPQ fluorescence by commonly used buffers was investigated as a function of
pH. Titration curves demonstrated SPQ quenching and yielded pK values
characteristic of each buffer. from here, pH-independent Stern-Volmer
constants, KQbase, were calculated for each of the morpholine derivatives,
MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES
and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine
derivatives, exhibited an additional pH-independent "molecular" quenching
constant KmQ throughout the pH range 3-10. To study chloride fluxes,
therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl,
which is a function of both pH and buffer composition. Equations describing
these relationships are presented. In conclusion, unless both pH and the
buffer composition are taken into account, SPQ is unsuitable for studying
the concomitant transmembrane fluxes of Cl- and H+.
