The isoelectric point (pI) is the pH at which a molecule carries no net electric charge. For amino acids and proteins it is one of the most useful single numbers: at its pI a protein does not move in an electric field, it is usually least soluble, and it binds to neither cation- nor anion-exchange resins. Isoelectric focusing, ion-exchange chromatography and protein crystallisation are all planned around it.
For a simple amino acid with one acidic and one basic group, the pI is just the average of the two pKa values – glycine has pKa values of 2.34 and 9.60 and a pI of 5.97. Amino acids with an ionisable side chain have three pKa values, and the pI is the average of the two that bracket the neutral zwitterion: the two lowest for aspartate and glutamate, the two highest for lysine, arginine and histidine.
Peptides and proteins contain many ionisable groups – the N- and C-termini plus every Asp, Glu, Cys, Tyr, His, Lys and Arg side chain. Their net charge at any pH follows from the Henderson–Hasselbalch equation, and the pI is the pH where the sum is zero, found numerically. Published pKa sets differ, so this calculator lets you choose between the EMBOSS set and the Lehninger textbook set and shows how much the answer depends on that choice.
How to use the Isoelectric Point Calculator
- Calculate pI from
- pKa₁ (acidic group, e.g. COOH)
- pKa₂ (basic group, e.g. NH₃⁺)
- Isoelectric point pI
- Amino acid
- Sequence (one-letter code) — Spaces and numbers are ignored, e.g. DRVYIHPF (angiotensin II)
- pKa set
- pH for the net charge
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How it works
In the pKa mode the two values are averaged; the reverse mode solves for the missing pKa. For a single amino acid the calculator sorts its pKa values and averages the two either side of the zero-charge form. For a sequence it counts each ionisable residue, adds up the Henderson–Hasselbalch charges of all groups and bisects between pH 0 and 14 until the net charge is zero.
Worked example
Single amino acid. Lysine has pKa values of 2.18 (α-COOH), 8.95 (α-NH₃⁺) and 10.53 (side-chain NH₃⁺). Fully protonated it carries +2, so the neutral form lies between the second and third ionisations: pI = (8.95 + 10.53) / 2 = 9.74.
Peptide. Angiotensin II, DRVYIHPF, has an N-terminus, a C-terminus, one Asp, one Arg, one His and one Tyr. With the EMBOSS pKa set (N-term 8.6, C-term 3.6, D 3.9, R 12.5, H 6.5, Y 10.1) the net charge is zero at pH 7.54; with the Lehninger set it is 7.76. At pH 7 the net charge is +0.22 (EMBOSS). Its formula is C₅₀H₇₁N₁₃O₁₂, average molecular weight 1 046.2 g/mol.
pKa values and pI of selected amino acids (Lehninger)
| Amino acid | pKa₁ (COOH) | pKa₂ (NH₃⁺) | pKa R | pI |
|---|---|---|---|---|
| Glycine | 2.34 | 9.60 | – | 5.97 |
| Alanine | 2.34 | 9.69 | – | 6.01 |
| Valine | 2.32 | 9.62 | – | 5.97 |
| Leucine | 2.36 | 9.60 | – | 5.98 |
| Serine | 2.21 | 9.15 | – | 5.68 |
| Cysteine | 1.96 | 10.28 | 8.18 | 5.07 |
| Tyrosine | 2.20 | 9.11 | 10.07 | 5.66 |
| Histidine | 1.82 | 9.17 | 6.00 | 7.59 |
| Lysine | 2.18 | 8.95 | 10.53 | 9.74 |
| Arginine | 2.17 | 9.04 | 12.48 | 10.76 |
| Aspartate | 1.88 | 9.60 | 3.65 | 2.77 |
| Glutamate | 2.19 | 9.67 | 4.25 | 3.22 |
pKa sets used for peptides
| Group | EMBOSS | Lehninger |
|---|---|---|
| N-terminus | 8.6 | 9.69 |
| C-terminus | 3.6 | 2.34 |
| Asp (D) | 3.9 | 3.86 |
| Glu (E) | 4.1 | 4.25 |
| Cys (C) | 8.5 | 8.33 |
| Tyr (Y) | 10.1 | 10.0 |
| His (H) | 6.5 | 6.0 |
| Lys (K) | 10.8 | 10.5 |
| Arg (R) | 12.5 | 12.4 |
Tips
- Keep enzyme and substrate concentrations in the same unit.
- Check that your data lie in the linear range of the standard curve.
- pH strongly affects enzyme activity and protein solubility.
- Everything is calculated in your browser.
Common mistakes to avoid
- Extrapolating a calibration curve far beyond the standards.
- Mixing units (U, kat, µmol/min).
- Ignoring dilution factors when reporting the original concentration.
Formula
pI = (pKa₁ + pKa₂) / 2 (the two pKa values either side of the neutral form) · peptides: Σ basic nᵢ/(1 + 10^(pH − pKaᵢ)) − Σ acidic nⱼ/(1 + 10^(pKaⱼ − pH)) = 0
Worked example
Inputs — Calculate pI from: A peptide or protein sequence; Sequence (one-letter code): DRVYIHPF; pKa set: EMBOSS; pH for the net charge: 7.
Results — Isoelectric point (pI) = 7.54; Missing pKa = 9.6; Net charge at the chosen pH = 0.216; Number of residues = 8; Average molecular weight = 1,046.2 g/mol; Molecular formula (free termini, reduced cysteines) = C₅₀H₇₁N₁₃O₁₂.
Key terms
- Isoelectric point (pI)
- pH at which a molecule has zero net charge.
- Zwitterion
- A molecule with equal positive and negative charges, e.g. ⁺H₃N–CH₂–COO⁻ for glycine near neutral pH.
- pKa
- −log₁₀ of the acid dissociation constant; the pH at which a group is half protonated.
- Henderson–Hasselbalch equation
- pH = pKa + log([A⁻]/[HA]), used to work out the fraction of each group that is charged.
Frequently asked questions
How do you calculate the isoelectric point of an amino acid?
Average the two pKa values on either side of the neutral (zwitterion) form. For glycine, (2.34 + 9.60) / 2 = 5.97.
How do you find the pI of an amino acid with three pKa values?
For acidic side chains average the two lowest pKa values (aspartate: (1.88 + 3.65) / 2 = 2.77); for basic side chains average the two highest (lysine: (8.95 + 10.53) / 2 = 9.74).
How is the pI of a protein calculated?
Count the ionisable groups (termini, D, E, C, Y, H, K, R), write the Henderson–Hasselbalch charge of each as a function of pH and find the pH where the total is zero. Computers do this by bisection, as this calculator does.
Why do different pI calculators give different answers?
They use different pKa sets. For angiotensin II (DRVYIHPF) the EMBOSS set gives 7.54 and the Lehninger set 7.76. Measured pI values can differ further because folding shifts pKa values.
What is the charge of a protein above and below its pI?
Below the pI it is positively charged (binds cation exchangers); above the pI it is negatively charged (binds anion exchangers). At pH 7 a protein with pI 5 carries a net negative charge.
Why are proteins least soluble at their pI?
With no net charge there is no electrostatic repulsion between molecules, so they aggregate and precipitate more easily – the basis of isoelectric precipitation of casein at about pH 4.6.
Which amino acid has the highest pI?
Arginine, at 10.76, because its guanidinium side chain (pKa 12.48) stays protonated until very high pH. Aspartate has the lowest, 2.77.
Which pKa set should I use?
Either is reasonable for an estimate. EMBOSS is the default of a widely used bioinformatics package; the Lehninger set comes from the standard biochemistry textbook. Report which one you used.