A novel method for measurement of intravesicular pH using fluorescent probes

A method for the measurement of intravesicular pH of phospholipid vesicles and gastric microsomes is described. The present method makes use of the well characterized pH-dependent shift of the emission maximum of two fluorescent amines, quinine and acridine. As the probes distribute into the intravesicular space, according to the existing pH gradient, they respond to the acidic environment and emit fluorescence at a longer wavelength than the external probes which sense the more alkaline medium. By measuring both the decrease in the alkaline fluorescence peak and the enhancement of the acidic peak, direct determination of the internal pH can be obtained. This method has the advantages of giving a positive signal (enhancement of fluorescence instead of quenching), measuring intravesicular pH directly without the need of independent measurement of the volume of the H+ space, and also enabling the possible use of a much lower probe concentration than that required by the fluorescence-quenching method. The accuracy of the present method was quantitatively verified using phospholipid vesicles as a well defined model system. Application to biological systems was demonstrated using gastric microsomes which actively transport H+ into the microsomal space via an H+-K+ exchange ATPase system. The reasoning of the present method is also extended to the monitoring of internal alkaline pH gradient by using a fluorescent weak acid, o-hydroxycinnamic acid. Some general criteria for the future search for better Ph gradient probes are presented.