Isoelectric Point Calculator: pI and Net Charge

What is the isoelectric point?

The isoelectric point, written pI, is the pH at which a molecule has no net electrical charge. Amino acids and peptides carry both acidic groups (carboxyls, and some side chains) and basic groups (amines, and some side chains). Each group gains or loses a proton depending on the pH. At low pH most groups hold a proton, so the molecule is positive. At high pH most have lost one, so it is negative. The pI is the crossover point.

At its pI a molecule does not move in an electric field. Above its pI it carries a net negative charge and moves toward the positive electrode (anode). Below its pI it is positive and moves toward the negative electrode (cathode). A free amino acid at its pI exists mostly as a zwitterion: one molecule that carries a positive and a negative charge at the same time.

How to use the isoelectric point calculator

1. Paste the sequence in one-letter code or hyphenated three-letter code. The calculator strips spaces, numbers and FASTA headers, reads Ac- and -NH₂ written at the ends, and flags any letter that is not a standard amino acid.
2. Choose a pKa set. IPC_peptide is the default because it performed best on measured peptide pI values. Choose IPC_protein for full proteins, or Bjellqvist to reproduce ExPASy.
3. Set the pH for the net-charge readout. The calculator also always shows the charge at pH 7.4.
4. Set the ends and disulfides. Acetyl and amide caps remove a terminal charge. Cysteines locked in disulfide bonds cannot lose a proton, so they drop out of the sum.
5. Read the results. The large number is the pI. Below it you will find the charge at your pH, the number of basic and acidic groups, the charge curve, the pI from every pKa set and, under Show the math, each group's contribution.

The Henderson–Hasselbalch formulas for net charge

The Henderson–Hasselbalch equation links pH, pKa and the fraction of a group that is protonated. The pKa is the pH at which a group is half protonated. From it, each group's average charge at any pH follows directly.

Values from Kozlowski 2016. Other sets are compared further down.

How to find the isoelectric point of an amino acid

A free amino acid has two or three ionizable groups, each with its own pKa. The shortcut: start from the fully protonated form, remove protons in order of increasing pKa, and stop at the form with zero net charge. The pI is the average of the two pKa values on either side of that form.

Glycine: two groups

Glycine's α-carboxyl has pK₁ = 2.34 and its α-amine has pK₂ = 9.60. Fully protonated glycine is +1. Losing the carboxyl proton gives the neutral zwitterion. So pI = (2.34 + 9.60) ÷ 2 = 5.97. At that pH only 0.023% of molecules still carry a neutral COOH and the same 0.023% carry a neutral NH₂, so the charges cancel exactly.

Aspartic acid and lysine: three groups

Aspartic acid has pKa values of 1.88, 3.65 (side-chain carboxyl) and 9.60. Fully protonated it is +1, and it becomes neutral after losing its first proton, so the pI averages the two lowest values: (1.88 + 3.65) ÷ 2 = 2.77. Lysine starts at +2 because its side-chain amine is also protonated. It is neutral after losing two protons, so the pI averages the two highest values: (8.95 + 10.53) ÷ 2 = 9.74.

Isoelectric points of the 20 amino acids

The pKa values below are the classic set for free amino acids at 25 °C, as printed in OpenStax Organic Chemistry Table 26.1. The pI column is computed from them with the averaging rule, so you can check every row by hand.

Computed pI values can differ from printed tables by 0.01 because of rounding. Tryptophan’s pK₁ is 2.38 (Dawson, Data for Biochemical Research); OpenStax prints 2.83, which does not reproduce its own pI of 5.89. OpenStax lists 6.02 for isoleucine, while its printed pKa values average to 5.98.

The averaging rule assumes the other groups are fully on or off at the pI. That holds when pKa values are far apart. Tyrosine is the exception: its side-chain pK_R (10.07) sits close to pK₂ (9.11). Solving the full Henderson–Hasselbalch sum gives 5.63, not 5.66. For peptides, where groups crowd together, the calculator always solves the full sum.

How to calculate the isoelectric point of a peptide by hand

Peptides lose their internal α-amines and α-carboxyls to peptide bonds. Only the two termini and the side chains can ionize. Use GHK (Gly-His-Lys) and the IPC_peptide pKa values as an example.

1. List the ionizable groups. N-terminus (basic, pKa 9.564), Lys (basic, pKa 10.517), His (basic, pKa 6.018), C-terminus (acidic, pKa 2.383).
2. Find the starting charge. At very low pH all three basic groups are protonated and the C-terminus is neutral: +3.
3. Remove protons in pKa order. Past pH 2.383 the C-terminus goes to −1 (net +2). Past 6.018 histidine loses its proton (+1). Past 9.564 the N-terminus loses its proton (0). Past 10.517 lysine does (−1).
4. Average the pKa values that bracket zero. (9.564 + 10.517) ÷ 2 = 10.04.
5. Check with the full sum. The calculator’s bisection gives 10.04. The shortcut works here because the neighboring pKa values sit well apart.

The bisection method is simple. Start with pH 0 and pH 14, where the charge is surely positive and negative. Test the midpoint. If the charge there is positive, the pI lies above it; if negative, below. Halve the interval and repeat. Fifteen halvings pin the pI to better than 0.001 pH unit, which is the approach Kozlowski describes for IPC.

Net charge vs pH

The charge curve shows the whole story at once. Each ionizable group contributes an S-shaped step centred on its pKa. The curve for angiotensin II crosses zero at its pI of 7.73. Near neutral pH it runs almost flat, so the charge at pH 7.4 is only +0.03.

The table gives the same information as numbers for three example peptides.

Why pKa sets give different answers

A pKa is not a fixed constant. It shifts with neighboring residues, temperature, ionic strength, denaturants and whether the group is buried in a folded protein. Each published set was measured or fitted under different conditions, so each gives a somewhat different pI. The calculator includes seven sets from the IPC comparison by Kozlowski (2016).

Bjellqvist also changes the N-terminal pKa for seven N-terminal residues (A, M, S, P, T, V, E), as ExPASy does.

Which set should you trust? On a benchmark of peptides with measured pI values, Kozlowski found IPC_peptide had the lowest error (RMSD 0.251), closely followed by Solomon (0.255) and Lehninger (0.262). EMBOSS scored 0.325 and Bjellqvist 0.669. The Bjellqvist set, used by ExPASy, was derived from protein migration in immobilized pH gradient gels containing 9.2–9.8 M urea, which suits denatured proteins better than short peptides. Our Bjellqvist results match ExPASy Compute pI/Mw to the second decimal.

Why pI matters: solubility, IEF and ion exchange

Solubility is lowest near the pI

With no net charge, molecules repel each other least and are most likely to aggregate or precipitate. Shaw and colleagues showed this directly: as they swapped acidic residues of ribonuclease Sa for lysines, the pH of lowest solubility moved with the pI. If a peptide will not dissolve, a solvent whose pH sits further from the pI often helps. Follow the product’s instructions first. Once it is in solution, the peptide reconstitution calculator and the BAC water calculator handle concentration and volume, and the mcg to mg converter handles unit changes.

Isoelectric focusing

In isoelectric focusing (IEF), molecules move through a pH gradient until they reach the pH equal to their pI, where they stop. It is the first dimension of 2-D gel electrophoresis and of capillary IEF used with mass spectrometry. A predicted pI tells you where to look.

Ion exchange chromatography

Below its pI a peptide is positive and binds a cation exchanger. Above its pI it is negative and binds an anion exchanger. The farther the buffer pH is from the pI, the larger the net charge, which usually means stronger binding.

Worked examples

Angiotensin II, DRVYIHPF

Basic groups: N-terminus, Arg and His. Acidic groups: C-terminus, Asp and Tyr. With IPC_peptide the pI is 7.73. Across sets it ranges from 6.74 (Bjellqvist) to 7.76 (Lehninger), a wide spread. The pI falls between His and the N-terminus, and the N-terminal pKa alone ranges from 7.50 to 9.69 across sets.

BPC-157, GEPPPGKPADDAGLV

One Lys and the N-terminus face three acidic side chains (Glu and two Asp) plus the C-terminus. The pI is 3.74, and the net charge at pH 7.4 is −2.01. Its average molecular weight is on the peptide molecular weight calculator. BPC-157 is an investigational peptide, not an FDA-approved drug; the BPC-157 calculator covers its status and vial math.

Oxytocin, amidated with one disulfide

Oxytocin (CYIQNCPLG-NH₂) has an amidated C-terminus and a disulfide between its two cysteines. That leaves only the N-terminus (basic) and Tyr (acidic) to ionize, so the pI is 9.82 and the charge at pH 7.4 is +0.99. Entered with both cysteines free, the pI would drop to 8.24, which is why the disulfide input matters.

Capping changes everything

Acetylating angiotensin II removes one positive charge and drops the pI from 7.73 to 4.96. Amidating it instead removes a negative charge and raises the pI to 9.82. A fully capped peptide such as Ac-GHK-NH₂ has no acidic group at all, so it is positive at every pH and has no pI; the calculator says so instead of printing a number.

Common mistakes

• Letters that are not amino acids. B, J, O, U, X and Z are ignored, with a red warning. Check that the remaining length is right.
• Three-letter code in capitals without hyphens. Gly-His-Lys is read as GHK, but GLYHISLYS is read as nine one-letter residues. An empty box gives no result.
• Ignoring disulfides. Cysteines in disulfide bonds cannot ionize. Enter the bonds, and never more than half the Cys count; the calculator caps it and warns you.
• Forgetting caps. Acetyl and amide groups each remove a terminal charge and can move the pI by several units.
• Mixing pKa sets. Compare peptides with the same set. A difference of 0.3 between two tools may just be the pKa values.
• Treating the pI as exact. It is a prediction. Phosphorylation, deamidation, metal binding and folding all shift the real value.
• Treating a blend as one peptide. A vial with two peptides holds two molecules with two pIs. Calculate each sequence separately; the peptide blend calculator splits the amounts.
• Typing a pH outside 0–14. The calculator falls back to pH 7 for the charge readout and shows a warning.

Key terms

Isoelectric point (pI)

The pH at which a molecule’s net charge is zero.

pKa

The pH at which an ionizable group is half protonated.

Henderson–Hasselbalch equation

pH = pKa + log([base] ÷ [acid]); used here to find each group’s fractional charge.

Zwitterion

A molecule that carries both a positive and a negative charge but has zero net charge.

Net charge

The sum of all positive and negative partial charges at a given pH.

Bisection

A search that halves the pH interval until the charge is zero within the chosen precision.

Isoelectric focusing (IEF)

Electrophoresis in a pH gradient that separates molecules by pI.

Cystine

Two cysteines joined by a disulfide bond. Its sulfur atoms carry no ionizable proton.

Frequently asked questions

Sources

We check every formula and figure on this page against primary sources. See our editorial policy for how we review content.