Vinicius Waldow : There seems to be a misunderstanding. Phosphoric acid H3PO4 (or, perhaps clearer, O=P(OH)3) can stepwise give up three protons, each of these deprotonations has its own pKa value: H3PO4 H2PO4- HPO42- PO43-, with pKa 2.15, 7.20 and 12.33, respectively, at 25 °C. In nucleic acids, two of the three OH-groups are used up for bonds to (desoxy)ribose, only one acidic group is left. Because of mesomery between the =O and -O- groups, the anion is stabilised and the pKa correspondingly low, some sources say 2, some even 0. Regardless of the exact value, all phosphate in nucleic acids are fully deprotonised at cellular pH.

According to this source, the pKa of the phosphate in nucleic acid phosphodiester bonds is near zero. Hence, the groups are fully ionized at neutral pH.

https://en.wikibooks.org/wiki/Structural_Biochemistry/Nucleic_Acid/DNA/DNA_structure

The pKa of phosphate groups in DNA or RNA is 2 and gives a negative charge at neutral pH (pH=7). This charge-charge repulsion forces the phosphate groups to take opposite positions of the DNA strands and is neutralized by proteins (histones), metal ions such as magnesium and polyamines. See this lecture note I got from Harvard.

Vinicius Waldow :

In order to understand the pKa of the phosphate groups in DNA, the Henderson-Hasselbalch equation and the intracellular pH must be taken into account.

The intracellular pH is 7.4 (approximately), so the pKa of the phosphate group (cellularly relevant) is 7.2.

If you apply the equation, you will find that a phosphate group will be found in the third form (i.e., with two deprotonated hydroxyl groups) for the most part (61.24%).

Now, it is important to consider that the phosphate groups in DNA come from dNTPs. So you have to look at how this molecule comes about.

I leave an image with the application of the equation and the proportion of each form of the phosphate group that would remain in the intracellular medium. I hope this helps you to be clearer about your question.

Does the microenvironment perturb the normal pKa value of 2 or not ?

Vipul Batra :

The pKa value is a constant inherent in each chemical species. So, changes in the microenvironment would affect the ratio of the protoned and deprotoned species pair, but not the pKa value.

I hope I understood your question.

Vinicius Waldow : There seems to be a misunderstanding. Phosphoric acid H3PO4 (or, perhaps clearer, O=P(OH)3) can stepwise give up three protons, each of these deprotonations has its own pKa value: H3PO4 H2PO4- HPO42- PO43-, with pKa 2.15, 7.20 and 12.33, respectively, at 25 °C. In nucleic acids, two of the three OH-groups are used up for bonds to (desoxy)ribose, only one acidic group is left. Because of mesomery between the =O and -O- groups, the anion is stabilised and the pKa correspondingly low, some sources say 2, some even 0. Regardless of the exact value, all phosphate in nucleic acids are fully deprotonised at cellular pH.

Perturbed or apparent pKa??

My Bible (Nelson & Cox) says,"The phosphate groups, ,with a pKa near 0, are completely ionized and negatively charged at pH 7 and the negative charges are generally neutralized by ionic interactions with positive charges on proteins, polyamines metal ions, and polyamines."

Adam B Shapiro and Vipul Batra , thank you for the references.

Engelbert Buxbaum, I just pointed out in my question about the unavailability of two hydroxyl groups from phosphates due to their participation in the phosphodiester bond. My question was exactly if the pKa of the hydroxyl group of phosphate bonded to DNA or RNA that is available was different from the lowest pKa of the free phosphoric acid. Anyway, thanks for the more precise values of pKa1, pKa2 and pKa3 for the free phosphoric acid.

Maurice Ekpenyong, thanks for the explanation, but my question was about phosphates bonded in DNA and RNA molecules, where each phosphate have only one hydroxyl group that can pronotate and deprotonate. (The exception are phosphates at the 5'-end, that have two hydroxyl groups.)

It seems that we don't have a more precise value than "something between 0 and 2" for this pKa. I was wondering if there was a study that tried to directly measure this value, but I couldn't find.

A figure to illustrate phosphate groups in nucleic acids.

I have an interesting thought: certain hot springs bacteria live quite comfortably in pH = 1 environments. Would not their DNA be significantly less ionized? If so, since magnesium would not be coordinated to the phosphates of the backbone, what do they do to maintain DNA structure? It can't be the same as bacteria living in a more neutral or alkaline environment, but evolutionary patterns suggest it cannot be that different either. Does anyone know?

Benjamin Lasseter : Even most acidophils have a cytosolic pH near neutral, they prevent entry of protons into the cytosol and have pumps to remove them against a concentration gradient (see for example http://biology.kenyon.edu/slonc/Micro/Cyto_pH_Ch1.pdf). There are exceptions, however (doi:10.1007/BF00414767 )

Thanks. That makes sense, it being a simple solution to the problem, using a motif I am very familiar with. It's obviously an off day for me, or I would have seen that.

I just found the pKa value of dimethylphosphoric acid (CH3-O-)2P(=O)OH as 1.54 (doi:10.1021/ja01252a028, https://www.chem.wisc.edu/areas/reich/pkatable/pKa_compilation-1-Williams.pdf), the pKa increases slightly with alkyl chain length. Unfortunately, I could find nothing about (HO-CH2-)2P(=O)-OH, which might be the even more relevant model compound.