Average the two pKa values that bracket the neutral form
At the isoelectric point the net charge is zero. For a small molecule that happens midway between the two ionizations on either side of the neutral (zwitterionic) form, so the pI is simply their average.
The trick is choosing the right pair. Use the two pKa values that sit directly above and below the form with no net charge, not just the highest and lowest.
Which pKa pair to use
| Amino acid type | Average these |
|---|---|
| No charged side chain (e.g. Gly, Ala) | carboxyl pKa + amino pKa |
| Acidic side chain (Asp, Glu) | carboxyl pKa + side-chain pKa |
| Basic side chain (His, Lys, Arg) | amino pKa + side-chain pKa |
Typical values: carboxyl ~2.3, amino ~9.6. An acidic side chain pulls the pI down toward 3; a basic one pushes it up toward 10.
What the pI tells you
- Below the pI the molecule is net positive and drifts toward the cathode in electrophoresis; above the pI it is net negative and moves toward the anode.
- At the pI it carries no net charge, does not migrate, and usually reaches its lowest solubility, which is why proteins tend to precipitate there.
- Proteins are different. With many ionizable groups there is no simple average; you solve for the pH where total charge is zero. The two-pKa average applies to single amino acids and simple peptides.
Common questions
What is the isoelectric point?
It is the pH at which a molecule carries no net electric charge, so positive and negative groups exactly cancel. At this pH the molecule does not migrate in an electric field and is often least soluble.
How do you calculate the pI of an amino acid?
Average the two pKa values that flank the neutral form. For an amino acid with no charged side chain, that is the carboxyl pKa and the amino pKa. For one with a charged side chain, use the two pKa values on either side of the zwitterion.
Which pKa values do I average for an acidic vs a basic side chain?
For an acidic side chain (Asp, Glu) average the carboxyl and side-chain pKa, both low. For a basic side chain (His, Lys, Arg) average the amino and side-chain pKa, both high. That pins the pI to the pair that brackets the neutral species.
