Practical Considerations
Using the pH probe in the O2k to measure proton production requires some modifications of standard protocols and in fact a certain degree of method development. Some of the encountered challenges are
- very small buffering capacity required
- determination of buffering capacity necessary to calculate real proton flux
One approach we are considering now is to use the TIP to run in a "pH stat"mode, i.e. keeping the pH constant by a feedback controlled automatic injection of base, then determining proton flow from the amount of base injected. This circumvents the determination of buffering capacity. The "pH-Stat" approach might also make buffering obsolete. Alternately, the proton flow can be determined by
- injecting acid into the medium (without any biological sample) thereby determining the buffering capacity,
- running your experiment
- converting the logarithmic change in pH to a linearized Proton concentrations
- considering the buffering capacity determined before.
Potential Applications
On the simultaneous measurement of O2 and pH, we may refer to the classical literature on bioenergetics and the discovery of the chemiosmotic coupling mechanism, the quantification of H+/O2 stoichiometric ratios for proton pumping (Peter Mitchell). Other groups (e.g. Eskil Elmer - http://www.oroboros.at/index.php?id=mipnet-sweden#c1588) have used the pH electrode in the O2k in conjunction with a study of mitochondrial permeability transition.
The majority of novel applications will address the problem of aerobic glycolysis in intact cells, using the measurement of proton production as an indirect but continuous record of lactate production and corresponding acidification of the medium, while simultaneously monitoring oxygen concentration and oxygen consumption. In a well buffered culture medium, the pH change is extremely small relative to the amount of protons (lactic acid) produced, hence a low-buffering capacity medium needs to be applied. A titration of acid (lactic acid or HCl) into the low-buffering capacity medium yields the pH-dependent buffering capacity (Delta H+ added/Delta H+ measured by the pH electrode). Under various metabolic conditions, lactic acid production is the dominant mechanism causing acidification, hence the pH measurement is a good indirect indicator of aerobic glycolysis.
