# diprotonated vs monoprotonated inorganic phosphate

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?

Do the two buffering systems, bicarbonate vs HEPES, differ in their making available diprotonated vs monoprotonated inorganic phosphate at $$\mathrm{pH \ 7.4}$$, room temperature?

I do not have any physiology lab experience. However, my general knowledge is telling me that you have not completely disclosed what physiological response you are looking for and what components in the buffer would make that response (it is true that you have said that the study rely upon the transport of diprotonated inorganic phosphate, but should the cell enzymes taking monoprotonated inorganic phosphate and converting it to diprotonated inorganic phosphate?). If we know those little details, if would have been easy to answer your question.

Well, $$\mathrm{p}K_\mathrm{a2}$$ of HEPES buffer is 7.5 at $$\pu{25 ^\circ C}$$ while that of diprotonated phosphate $$(\ce{H2PO4-})$$ is 7.2 at $$\pu{25 ^\circ C}$$. Therefore, at $$\mathrm{pH} \ 7.4$$, monoprotonated/diprotonated phosphate ions would exists in HEPES buffer, if you added some $$(\ce{NaH2PO4})$$ to the buffer. However, the amount of monoprotonated phosphate $$(\ce{HPO4^2-})$$ would be more compared to that of diprotonated phosphate ions $$(\ce{H2PO4-})$$ in HEPES buffer $$(\mathrm{p}K_\mathrm{a2, \ HEPES} = 7.5)$$ during your physiological response checking.

On the other hand, $$\mathrm{p}K_\mathrm{a}$$ of carbonic acid is 6.1. The bicarbonate buffer system is an acid-base homeostatic mechanism involving the balance of carbonic acid $$(\ce{H2CO3})$$, bicarbonate ion $$(\ce{HCO3-})$$, and carbon dioxide $$(\ce{CO2})$$ in order to maintain $$\mathrm{pH}$$ in the blood and duodenum, among other tissues, to support proper metabolic function. That's why you need to maintain the partial pressure of $$\ce{CO2}$$ in the buffer system when you are using bicarbonate buffer. What I understand according to the process you have described in the question is when you added $$\ce{NaH2PO4}$$, $$\mathrm{pH}$$ might move from 7.4 towards 7.2 giving equal or higher amount of $$\ce{NaH2PO4}$$ compared to that of $$\ce{Na2HPO4}$$ $$(\mathrm{p}K_\mathrm{a, \ \ce{H2CO3}} = 6.1)$$ during your physiological response checking.

Although you get better response in bicarbonate buffer compared to that in HEPES buffer in this particular case probably based on the reasons given above, keep in mind that HEPES is widely used in cell culture when compared to bicarbonate buffers (which are also commonly used in cell culture), largely because it is better at maintaining physiological $$\mathrm{pH}$$ despite changes in carbon dioxide concentration, usually produced by aerobic respiration (Ref.1 & 2). I have attached the PDF of Ref.2 so that you can read and learn an specific case of comparison of HEAPS and bicarbonate.

References:

1. Simona C. Baicu and Michael J. Taylor, "Acid–base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency," Cryobiology 2002, 45(1), 33-48 (DOI: https://doi.org/10.1016/S0011-2240(02)00104-9).
2. Dayle H. Geroski, Roberts Grosserode, and Henry F. Edelhauser, "A Comparison of HEPES and Bicarbonate Buffered Intraocular Irrigating Solutions: Effects on Endothelial Function in Human and Rabbit Corneas," Journal of Toxicology: Cutaneous and Ocular Toxicology 1982-83, 1(4), 299-309 (DOI: https://doi.org/10.3109/15569528209079878)(PDF).