Determination of intracellular buffer values after metabolic inhibition by fluoride and nitrilotriacetic acid

Non-bicarbonate buffer values have been analysed in homogenates of squid mantle muscle and toad gastrocnemius muscle using the PCO2 equilibration technique. Homogenate metabolism was inhibited by the addition of potassium fluoride and nitrilotriacetic acid. After an initial change in the concentration of organic phosphates a new metabolic equilibrium was achieved which was insensitive to pH changes during PCO2 variations. Inaccurate measurements of homogenate buffer values could, thereby, be prevented. The accumulation of inorganic phosphate during the initial metabolic shift caused an increase in homogenate buffer values. Levels of unbound inorganic phosphate, which are effective in buffering, were estimated by enzymatic analysis in homogenate ultrafiltrates. Based on the evaluation of apparent dissociation constants pK', and buffer values for fluoride and inorganic phosphate, the non-bicarbonate, non-phosphatei (i: inorganic) buffer value of the tissue was calculated. The level of free inorganic phosphate in the resting tissue was considered for an estimate of the intracellular non-bicarbonate buffer value under control conditions.