Electric charge of biomolecules,
as a function of pH and their isoelectric point

Essential concepts

1. Chemical equilibrium (law of mass action) and Le Châtelier's principle. (If you are not familiar with this, find some information; example)
2. Definition of isoelectric point:

   The isoelectric point (pI) or isoelectric pH (pH_I) is the pH for which the molecule has zero net charge. That is, it may have charged (ionised) groups, but the overall sum of all positive charges equals that of the negative charges.

3. pI is a constant property for each molecule. It depends on its chemical structure, on which ionisable groups it possesses.
4. pH is a property of the medium, the solution. Therefore, it is common to all molecules present. We set pH experimentally, for instance adding an acid or a base, or a buffer solution; it is not the result of ionisation of biomolecules in the sample.

Charge varies as a function of pH

Charge of any biomolecule depends on pH, since all its ionisable groups experience equilibria where protons are involved, like these:

• AH A⁻ + H⁺
• BH⁺ B + H⁺

As a consequence:

• When pH is low, there will be .. .. high low concentration of protons, and the equilibria will shift to the .. .. left right
  your answer
• The lower the pH is, the charge of the molecule (which will have multiple groups of type A and B) will be .. .. more positive less positive or .. .. more negative less negative
  

Simulation: (please slide the pH control)

The value of charge depends on the isoelectric point and the pH

As it has just been shown, pH determines the charge of the biomolecule; but which sign and magnitude does that charge have?

Our reference is that charge is zero when pH=pI (per definition of the isoelectric point)
Read again your answer to the previous question, that says The lower the pH is, charge of the molecule will be... and apply it now:

• when pH is lower than the pI of a molecule, this will have .. .. positive negative charge
• when pH is higher than the pI of a molecule, this will have .. .. positive negative charge

Comparing the charge of several molecules

When several different molecules are present together in a mixture, all of them will be subjected to the same pH. How does the charge of the diverse molecules differ? Understanding this is important, for example, for the possibilities of separating them using electrophoresis or ion exchange chromatography.

Let's suppose two proteins, P and Q, whose isoelectric points are pI_P=4.6 and pI_Q=8.7

• If pH of the solution is 6.8, the charge of both proteins will be:
  charge of P: .. .. positive negative charge of Q: .. .. positive negative
  (review your answers to the previous section in order to find out how to answer this one)
• If pH of the solution is 4.0, the net charge will be:
  charge of P: .. .. more positive positive negative more negative charge of Q: .. .. more positive positive negative more negative
• If pH of the solution is 9.2, the charge will be:
  charge of P: .. .. more positive positive negative more negative charge of Q: .. .. more positive positive negative more negative

Let's review this visually:

Note that, for any pH, charge of protein P (the one with lower pI) is always .. .. more positive more negative than charge of protein Q (higher pI), even though the sign and magnitude changes.

Calculation of the isoelectric point

Theoretical calculation of the value of isoelectric point for individual amino acids and for peptides (guided examples and problems to solve, with self-assessment).
(Sorry, that's not yet translated into English)