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| == Practical Considerations ==
| | #REDIRECT [[O2k-pH ISE-Module]] |
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| 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
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| * very small buffering capacity required
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| * determination of buffering capacity necessary to calculate real proton flux
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| 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
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| # injecting acid into the medium (without any biological sample) thereby determining the buffering capacity, | |
| # running your experiment
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| # converting the logarithmic change in pH to a linearized Proton concentrations
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| # considering the buffering capacity determined before.
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| == Potential Applications ==
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| On the simultaneous measurement of O2 and pH, we may refer to the
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| classical literature on bioenergetics and the discovery of the
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| chemiosmotic coupling mechanism, the quantification of H+/O2
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| stoichiometric ratios for proton pumping (Peter Mitchell). Other groups (e.g. Eskil Elmer -
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| http://www.oroboros.at/index.php?id=mipnet-sweden#c1588) have used the pH
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| electrode in the O2k in conjunction with a study of mitochondrial
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| permeability transition.
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| The majority of novel applications will address the problem of aerobic
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| glycolysis in intact cells, using the measurement of proton production as
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| an indirect but continuous record of lactate production and corresponding
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| acidification of the medium, while simultaneously monitoring oxygen
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| concentration and oxygen consumption. In a well buffered culture medium,
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| the pH change is extremely small relative to the amount of protons (lactic
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| acid) produced, hence a low-buffering capacity medium needs to be applied.
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| A titration of acid (lactic acid or HCl) into the low-buffering capacity
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| medium yields the pH-dependent buffering capacity (Delta H+ added/Delta H+
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| measured by the pH electrode). Under various metabolic conditions, lactic
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| acid production is the dominant mechanism causing acidification, hence the
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| pH measurement is a good indirect indicator of aerobic glycolysis.
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| {{#set:Technical service=pH}}
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| {{Technical service}}
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