I am not talking about ion exchange chromatography. I am talking about soluble proteins in solution which have a certain buffer capacity. When you add resin beads which deionize the solution (something that can be checked using a conductivity meter), the pH of the solution after this treatment is assumed to be equivalent to the pI of the protein. What I cannot picture right is what does the ion catching has to do with the in-solution pH. I assume that with an acidic protein, for example, the free cations sort of surround the negative charges of the protein molecules. With sequestration of the cations the solution protons would instead interact with the protein instead than with the water molecules. But why to the point of protein neutralization? I am not sure how to think about it since when I add some small amount of salt to a gelatin solution treated with beads, the pH does not go back to normal. Is it because of aggregation upon reaching the pI?
pH is an artifact of the sum of positive and negative ions in a solution. The constraint is always that the sum of positive and negative charges must be equal (electroneutrality). If you use a resin to adsorb ions from the solution (liberating H+ and OH- in the process), these will equilibrate to form water at the appropriate pH to satisfy any unbalanced charge that fails to be adsorbed. A protein has a mixture of both positive and negative ionizable residues. Assuming this is held back from bonding to the solid phase of the resin, but all the other ions can penetrate and get adsorbed to the solid phase, then you are left with enough H+ ions to reach electroneutrality. pH is just - log [H+].
Why to neutralization of the protein? See fun gen chem level (probably wrong) estimates below and attached. Short version: Without salts around, charges are determined by acid/base interactions (as you said in the original post). Existing charges on proteins discourage the formation of additional charged residues with same sign. They also encourage formation of charges of the opposite sign, which leads to an overall neutral protein.
I don't know how to respond to the last two sentences. I've never used this method.
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Energy of acid dissociation: dG=RTln(Q/K) where K=10^-pKa, Q=10^-pH*(A-/HA). The file has an array of pKas and pHs to show how different side chains will react as the pH changes.
Energy of charge/charge interactions: E=kqq/r The file shows distances ranging from 1-100 Angstroms, and major/minor/stokes radii of BSA for references.
An acidic residue (pKa=4) in water at pH 5 will release the proton (dG=-5.7 kJ/mol). If we add a negative charge 7 nm away (the length of the BSA elliptical major axis, AKA as far away as you can get on BSA) deprotonation of our acidic residue results in a positive (unfavorable) electronic interaction of 20 kJ/mol. Not until about pH 6.5 (dG=-20 kJ/mol) is there enough energy to overcome the unfavorable charge interactions. If instead we had a histidine residue (pKa=6), it would spontaneously protonate even at pH 9 (dG=+17 kJ/mol) in order to create attractive forces (favorable energy) with a negative charge on the opposite side of the protein (E= -20kJ/mol).
All these estimates assume A-/HA ratio of 1. You can change this parameter and see how it affects things in the attached file. I welcome any corrections/improvements as this may become a fun homework assignment someday.
For those of you interested here is a little Excel spreadsheet for the estimation of pH (using Solver) for any concentration of various ions. You can add additional buffers to the list as long as you copy the right pKa equations for species of similar type. You can even compensate for changing water concentration (e.g., concentrated buffers or solvent-aqueous systems).
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