We have made confounding observations in the physiology lab working with inorganic phosphate. The cell processes we study rely upon the transport of diprotonated inorganic phosphate. But we get different results whether the extracellular buffers are bicarbonate or HEPES. We wonder if the two buffering systems, bicarbonate vs HEPES, differ in their making available diprotonated vs monoprotonated inorganic phosphate.
If we buffer our solutions with bicarbonate (25mM), pH to 7.4, and maintain with 95% O2/5% CO2, we get expected physiological responses over a range of 0-5 mM inorganic phosphate (Pi), added in the form of sodium monobasic phosphate.
However, if we try adding sodium dibasic phosphate, it is supersaturated at ~>2 mM Pi and pH 7.4 and unuseable.
If we buffer our solutions with HEPES (20mM), pH to 7.4, and bubble with 100% O2, the solution looks fine but we do not get expected physiological responses over the range of 0-5 mM inorganic phosphate (Pi), added in the form of sodium monobasic phosphate.
Sodium dibasic phosphate also dissolves fine into the HEPES buffer at pH 7.4. However, we still do not get the expected physiological responses.
It seems as though there is no diprotonated inorganic phosphate in the HEPES buffer no matter how Pi was originally added to the solution.
Other confounding issues could include whether bicarbonate (does) or HEPES (some argue does not) get into the intracellular space. Before getting into that question, we'd like to know:
Do the two buffering systems, bicarbonate vs HEPES, differ in their making available diprotonated vs monoprotonated inorganic phosphate at pH 7.4, room temperature?
Many thanks in advance.