Intracellular pH Determination by a 31P-NMR Technique. The Second Dissociation Constant of Phosphoric Acid in a Biological System

Abstract

To evaluate the accuracy of pH determination by ³¹P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK′) value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hiickel equation. Ions (Na⁺, K⁺, Mg²⁺, and Ca²⁺) and salivary protein affected the pK′ value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK′ of frog muscle at 24°C.