Relationship between intracellular proton buffering capacity and intracellular pH.

In a recent publication the widely held view that the intracellular proton buffering power [defined as the amount of acid or base that has to be added to the cytosol to change the intracellular pH (pHi) by one pH unit] increases as the intracellular pH decreases, has been challenged, with the opposite relationship being proposed. In that publication, buffering was defined not in terms of pH change, but in terms of the change in proton concentration. The reason for this re-definition was the fear that the conventional analysis, using as it does a logarithmic function (pHi), could bias the outcome in favor of an increasing buffering power with decreasing pHi. The new system uses a "buffering co-efficient," defined as the number of protons necessary to be added to the cytosol to change the intracellular proton concentration by 1 mM. We report the use of both of these methods to analyze the relationship of pHi and buffering power, using human peripheral leucocytes loaded with the pH-sensitive fluorophore BCECF examined over a very wide range of pHi values (pHi 6.0 to 7.5). The most common method for pHi perturbation for the measurement of buffering is used, the rapid diffusion of ammonia across the cell membrane. In this study, analysis for both a bicarbonate-containing "open" system and for a Hepes-buffered "closed" system was performed.(ABSTRACT TRUNCATED AT 250 WORDS)