Isoelectric Point Calculator (protein or peptide pI from sequence, EMBOSS pKa set)

Understanding the isoelectric point calculator — concept and principle

This calculator estimates the isoelectric point (pI) of a protein or peptide from its amino-acid sequence, the pH at which its net charge is zero, and gives the net charge at a chosen pH. Biochemists use it to plan ion-exchange chromatography, isoelectric focusing, buffers and protein crystallisation.

Each ionisable group contributes a partial charge; the pI is where the positives and negatives cancel.

How it is calculated

Each basic group (N-terminus, Lys, Arg, His) contributes +1 ÷ (1 + 10^(pH − pKa))

Each acidic group (C-terminus, Asp, Glu, Cys, Tyr) contributes −1 ÷ (1 + 10^(pKa − pH))

Net charge = sum of all contributions; pI is the pH where the net charge is 0, found by bisection between pH 0 and 14

pKa set (EMBOSS iep): N-terminus 8.6, C-terminus 3.6, Lys 10.8, Arg 12.5, His 6.5, Asp 3.9, Glu 4.1, Cys 8.5, Tyr 10.1.

The principle

Ionisable side chains gain or lose protons depending on pH. The Henderson–Hasselbalch equation gives the fraction of each group that is charged: a group is half charged when pH equals its pKa. At low pH most groups carry protons and the protein is positive; at high pH they lose them and it becomes negative. The net charge falls steadily with pH, so there is exactly one pH where it crosses zero, the isoelectric point. There a protein has minimal electrostatic repulsion and is often least soluble.

Worked example

Default inputs: the 30-residue sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (the B chain of human insulin), pH 7.4.

Counts: 30 residues; basic K + R + H = 4; acidic D + E = 2; Cys + Tyr = 4.
At pH 7.4: N-terminus +0.941, Lys +1.000, Arg +1.000, two His +0.224; C-terminus −1.000, two Glu −1.999, two Cys −0.147, two Tyr −0.004.
Net charge at pH 7.4 = 0.01; at pH 7.0 = 0.4.
Bisection finds the zero crossing at pI = 7.42.

How to read the result

Below its pI a protein is positively charged and binds to cation exchangers; above it, it is negative and binds to anion exchangers. Proteins are often least soluble near their pI. Different pKa sets (Bjellqvist, Lehninger and others) give values that can differ by a few tenths, so compare pIs calculated with the same method.

Common mistakes

Including signal peptides or tags that are not in the final protein.
Expecting the calculated pI to match the folded protein exactly; buried or interacting groups shift pKa.
Ignoring disulfide bonds, which remove cysteine charges.
Comparing values from tools using different pKa tables.

Keep in mind: The estimate assumes independent groups with fixed pKa values. Modifications, folding, metal binding and disulfide bonds can shift the real pI.

Frequently asked questions

What is the isoelectric point?

The pH at which a molecule carries no net electric charge.

Why do tools give different pI values?

They use different pKa tables for the ionisable groups.

Does a protein precipitate at its pI?

Solubility is often lowest near the pI, though it depends on salt and the protein.

Are Cys and Tyr important?

Mainly at high pH, where their side chains begin to lose protons.

Sources: EMBOSS iep default pKa table, as listed in the MathWorks Bioinformatics Toolbox documentation for isoelectric

Checked 9 Nov 2026. pKa values: EMBOSS iep default table (Amino.pK), as listed in the MathWorks Bioinformatics Toolbox documentation for isoelectric (“EMBOSS amino acid pK table”). Charge model: Henderson–Hasselbalch equation applied to each ionisable group.

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