I want to know the protonation state of the intermembrane subunit of a protein.

For that, I need to know the pH, so given that small molecules can cross the lipid bilayer, could we assume intracellular and intermembrane pH is the same?

  • $\begingroup$ Not an answer, but perhaps supporting evidence for an answer. This article found significant pH differences among the mitrochondrial matrix, its intermembrane space, and the surrounding cytosol. While this is about the pH within an organelle membrane, it suggests that the cellular membranes will not necessarily have the same pH as the intracellular region. $\endgroup$
    – Tyberius
    Jul 7 at 19:55

Generally not. The pH difference between the two sides of a membrane can be very noticeable, for example when the membrane is that of a mitochondrion. Actually mitochondria works by actively transporting protons from one side of the membrane to the other side, using the chemical energy released by reducing O2 to H2O, and then use the proton gradient to generate ATP. If you have the same pH on both sides of your mitochondria membranes, you'll die instantly because of a lack of ATP, as will happen if you are poisoned by cyanide.

Note that only neutral, relatively hydrophobic small molecules can readily cross the lipid bilayer. H3O+ and OH- are both charged and extremely hydrophilic, so they don't pass through the lipid bilayer at all, that's why the pH of the two sides of a lipid bilayer don't spontaneously equilibrate. To let the pH equilibration to actually happen, you need a "shuttle" molecule, such that both the molecule itself and its conjugate acid/base are hydrophobic (for example, https://en.wikipedia.org/wiki/2,4-Dinitrophenol). The molecule can then carry protons through the membrane and do the equilibration.

If you are referring to the protonation states of the part of protein that is within the lipid bilayer (that is the transmembrane structural domains; they are of course dominated by hydrophobic residues, but a stray acidic/basic residue isn't entirely impossible), that's an even more difficult question, as that part of protein cannot readily equilibrate with either side of the membrane. I'm afraid that the protonation states of these residues are dependent on the history of the synthesis of the protein, and cannot be deduced from the protein structure itself. Perhaps someone else may be an expert on this topic and can give a better answer on this.


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