Peptide isoelectric point calculator
Paste a sequence and get its theoretical pI and net charge across the pH range.
What the isoelectric point is
The pH at which a peptide carries no net charge. Above it the molecule is net negative, below it net positive, and at it the positive and negative groups exactly cancel.
It is a practical number, not a trivia one. Net charge drives how a peptide behaves in ion-exchange chromatography and in electrophoresis, and solubility is usually at its lowest near the pI, where there is no net charge to keep molecules apart.
How it is calculated
Each ionisable group is treated with the Henderson-Hasselbalch relation: at a given pH, a basic group is protonated in a proportion set by the distance between that pH and the group's pKa, and an acidic group is deprotonated the same way. (Reference: Bjellqvist et al., 1994) The groups that matter are the two termini plus the side chains of lysine, arginine and histidine on the positive side, and aspartate, glutamate, cysteine and tyrosine on the negative.
Summing those contributions gives the net charge at any pH. The curve falls monotonically as pH rises, so the pI is found by bisection: halve the interval, look at the sign of the charge, keep the half that contains the crossing, repeat. The table above shows the same curve at seven points.
Why the pKa set has to be named
There is no single authoritative table of side-chain pKa values. Several sets have been published from different experimental work, and they disagree by a few tenths of a unit. Those differences propagate, so the same sequence can come out with a pI of 9.7 under one set and 9.9 under another, with neither being wrong.
This calculator uses the EMBOSS set and says so, which is the only thing that makes the number reproducible. A pI quoted with no set named cannot be checked against anything, the same way a purity figure with no method named cannot. Our note on what a purity figure measures makes the same point about chromatography.
What it does not account for
This is a theoretical pI from the sequence alone. It assumes every ionisable group behaves as if it were free in solution, which is a simplification: in a folded structure a charged residue can shift its neighbour's pKa substantially. It also ignores terminal modifications. An amidated C-terminus removes a negative charge and moves the pI up noticeably, and nothing in a letter sequence shows that it is there.
Frequently asked questions
What is the isoelectric point of a peptide?
The pH at which it carries no net charge, because its positive and negative groups exactly cancel. Above that pH the molecule is net negative and below it net positive.
Why do different calculators give different pI values?
Because there is no single agreed table of side-chain pKa values. Published sets disagree by a few tenths of a unit and that propagates, so the same sequence can come out at 9.7 under one set and 9.9 under another.
Which pKa set does this use?
The EMBOSS set, named on the page. A pI quoted without naming its pKa set cannot be reproduced or compared against anything.
Why does solubility drop near the pI?
Net charge is what keeps molecules apart. At the pI there is none, so the electrostatic repulsion that holds a peptide in solution is at its weakest and aggregation is most likely.
Does this account for C-terminal amidation?
No. Amidation removes a negative charge and moves the pI up noticeably, and nothing in a letter sequence shows that it is present. The same applies to N-terminal acetylation.
Is a theoretical pI the same as a measured one?
Not exactly. The calculation treats every ionisable group as if free in solution, whereas in a folded structure a charged residue can shift a neighbour pKa substantially. It is an estimate, and a useful one.