Isoelectric Point Calculator
Average two pKa values, never all three.
Work out Isoelectric Point. Average two pKa values, never all three. Names the misconception directly.
Isoelectric point
pI 5.97
Net charge is negative at physiological pH 7
With a neutral side chain the pI is the mean of the carboxyl and amino pKa values. Solubility is at its minimum at the pI, because the molecule carries no net charge to keep it apart from its neighbours — which is exactly how isoelectric precipitation separates proteins.
How the Isoelectric Point Calculator works
The pH at which an amino acid carries no net charge, from its carboxyl, amino and side-chain pKa values. Only the two pKa values that flank the neutral species are averaged — averaging all three is the standard error, and it moves the answer by several pH units.
Also known as: pi of an amino acid · net charge at ph 7 · which pka values do i average · isoelectric point of aspartate
Which two pKa values, and why not the third
The isoelectric point is the pH where the molecule carries no net charge. An amino acid passes through a sequence of species as pH rises, each one proton lighter than the last, and exactly one of them is neutral. The pI is the average of the two pKa values that bracket that neutral species — the one at which it appears and the one at which it disappears.
For a neutral side chain there are only two pKa values, so the question does not arise: glycine's pI is the mean of 2.34 and 9.60, giving 5.97. With three ionisable groups the choice matters, and picking wrong is the error this page exists to prevent.
Aspartate has pKa values of 1.88 (carboxyl), 3.65 (side-chain carboxyl) and 9.60 (amino). Its neutral form is bracketed by the two carboxyl values, so the pI is (1.88 + 3.65)/2 = 2.77. Averaging all three gives 5.04 — wrong by more than two pH units, which is enough to put a separation in entirely the wrong buffer.
Reading the sequence of species
At very low pH every ionisable group is protonated and the molecule carries its maximum positive charge. As pH rises, groups deprotonate in order of increasing pKa, each one removing a positive charge or adding a negative one. At very high pH everything is deprotonated and the charge is at its most negative.
The neutral species sits wherever that running total crosses zero. For an acidic side chain the molecule is already neutral after both carboxyls have gone, while the amino group is still protonated and positive — so the two carboxyl pKa values are the relevant pair, low down the scale. For a basic side chain the reverse holds and the pair sits high.
This is also how the net charge at any given pH is read off. Below the pI more of the ionisable groups are protonated than the neutral species requires, so the charge is positive; above it, negative. In electrophoresis that determines direction of travel, and at the pI there is no travel at all.
What the pI is used for
Solubility is at its minimum at the pI, because a molecule with no net charge has nothing to keep it apart from its neighbours. Isoelectric precipitation exploits this directly: shift the pH to a protein's pI and it drops out of solution while others stay dissolved. Casein precipitating out of souring milk is the same effect happening domestically.
Isoelectric focusing runs it as a separation. A pH gradient is set up across a gel and an electric field applied; each protein migrates until it reaches the pH equal to its own pI, where it stops moving because it has no charge left to push it. Resolution can reach 0.01 pH units, which is finer than most other protein separations manage.
For whole proteins, though, a pI calculated from a handful of pKa values is only an estimate. Hundreds of ionisable side chains contribute, and their pKa values are shifted by the local environment — a buried carboxyl can differ from the free amino acid value by several units. Sequence-based prediction software handles this; three numbers cannot.
Where to go next
The Isoelectric Point question rarely arrives on its own. These are the ones that usually come with it:
- pH Calculator — pH, pOH and both ion concentrations, on a log scale.
- Protein Concentration Calculator — A₂₈₀ to mg/mL, and when that stops working.
- Buffer Recipe Calculator — How much acid and base to weigh out for a target pH.
- Speed Distance Time Calculator — Any one of the three from the other two, in four units.
Frequently asked questions
How do I calculate the isoelectric point?
Average the two pKa values that flank the neutral form of the molecule. For a neutral side chain that is the carboxyl and amino values; with an ionisable side chain it is the side-chain pKa and whichever of the other two lies on the same side.
Why not average all three pKa values?
Because only two of them bracket the zwitterion. Averaging all three of aspartate's values gives about 5.5 instead of the correct 2.98 — a pH unit and a half of error, which is enough to ruin a separation.
What happens to a molecule at its pI?
It carries no net charge, so it does not migrate in an electric field and its solubility hits a minimum. Isoelectric precipitation and isoelectric focusing both exploit exactly this.
Which amino acids have unusual pI values?
The charged ones. Aspartate and glutamate sit near 3 because of their acidic side chains; lysine and arginine sit near 10 and 11 because of their basic ones. Most others cluster between 5.5 and 6.3.
What charge does an amino acid carry above its pI?
Negative. Above the pI the molecule has lost more protons than it has gained, so it migrates towards the anode in electrophoresis. Below the pI the reverse holds.
Does this work for whole proteins?
Only roughly. A protein's pI depends on hundreds of ionisable groups whose pKa values are shifted by the surrounding structure, so it is calculated from the sequence with software rather than from three numbers.
What is a zwitterion?
A molecule carrying both a positive and a negative charge with no net charge overall. Amino acids exist as zwitterions across most of the pH range — the carboxyl deprotonated and the amino protonated — which is why they are crystalline solids rather than the oily liquids their formulas suggest.
Why do amino acids have such high melting points?
Because they are zwitterionic, so the crystal is held by ionic attraction rather than by weaker intermolecular forces. Glycine melts with decomposition above 230 °C, far higher than any comparable neutral molecule of the same size.
How does the pI affect electrophoresis?
It sets the direction and speed of migration. Below its pI a molecule is positive and moves to the cathode; above it, negative and towards the anode. At the pI it does not move, which is what isoelectric focusing exploits to separate proteins to within 0.01 pH units.
Where does the pI of a protein come from?
The sum of every ionisable side chain plus the two termini, weighted by their local environments. A buried residue can have a pKa several units from its free amino acid value, so protein pI values are predicted from sequence with software rather than by averaging two numbers.
Which amino acid has the lowest pI?
Aspartate, at about 2.77, followed closely by glutamate at 3.22. Both have an acidic side chain that adds a low pKa to the pair being averaged. Arginine is highest at about 10.76.
Is the pI where a protein is least soluble?
Yes, because no net charge means nothing keeping molecules apart. Isoelectric precipitation uses this to separate proteins, and it is also why milk curdles as it sours — lactic acid drops the pH towards casein's pI near 4.6.
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