Short answer: The isoelectric point (pI) is the pH at which an amino acid or peptide carries, on average, no net electric charge: its positive and negative groups balance exactly [3]. Below its pI a molecule is mostly protonated and positively charged; above it, mostly deprotonated and negatively charged [2]. For a peptide, the pI comes from all of its ionizable groups together: the two termini plus any charged side chains [4].
This article is educational chemistry for laboratory readers. It does not describe biological effects, and it is not guidance for use in people or animals.
What the isoelectric point means
Amino acids carry both an acidic carboxyl group and a basic amino group, so the form they take in water depends on the pH:
- Zwitterions. In water, amino acids exist mainly as dipolar ions, or zwitterions, which are internal salts [1].
- Acid side vs base side. In acid solution an amino acid is protonated and exists mostly as a cation. In base it is deprotonated and exists mostly as an anion [2].
- The balance point. Between those extremes is a pH where the molecule is exactly balanced between anionic and cationic forms, existing mainly as the neutral zwitterion. That pH is the isoelectric point [2].
- Same idea for peptides. LibreTexts defines pI as the pH of an aqueous solution of an amino acid (or peptide) at which the molecules on average have no net charge [3]. Kozlowski's 2016 paper puts it as the pH at which a macromolecule's net charge is zero, so its electrophoretic mobility stops [4].
How to calculate pI for a single amino acid
The tool is the Henderson–Hasselbalch equation, pH = pKa + log([A⁻]/[HA]). When pH equals pKa, the two forms are present in equal amounts [2].
Worked example from OpenStax (alanine at pH 9.00): alanine's second pKa is 9.69, so log([A⁻]/[HA]) = 9.00 − 9.69 = −0.69. In a 1.00 M solution that works out to about 83% neutral zwitterion and 17% deprotonated anion [2].
The pI itself is the average of the two pKa values that involve the neutral zwitterion [2]:
- Neutral side chain: average pKa1 and pKa2. For alanine, (2.34 + 9.69) ÷ 2 ≈ 6.0. OpenStax lists 6.01 [1][2], and LibreTexts gives 6.02 [3].
- Acidic or weakly acidic side chain: average the two lowest pKa values [2]. For aspartic acid, (1.88 + 3.65) ÷ 2 ≈ 2.77, which matches the table value [1].
- Basic side chain: average the two highest pKa values [2]. For lysine, (8.95 + 10.53) ÷ 2 = 9.74. For arginine, (9.04 + 12.48) ÷ 2 = 10.76 [1].
Typical pI values of the 20 common amino acids
OpenStax tabulates pKa and pI values for all 20 [1]. The pattern:
- Neutral amino acids: the 15 neutral amino acids have pI values near neutrality, in the pH 5.0 to 6.5 range [2]. Examples: glycine 5.97, serine 5.68, proline 6.30 [1].
- Acidic amino acids: aspartic acid 2.77 and glutamic acid 3.22 [1].
- Basic amino acids: histidine 7.59, lysine 9.74 and arginine 10.76 [1].
- Weakly acidic side chains: cysteine (a thiol, pI 5.07) and tyrosine (a phenol, pI 5.66) are usually classed as neutral, but their side chains can be deprotonated in sufficiently basic solution [1].
From amino acid to peptide pI
For a peptide, the pI is set by every group that can carry a charge:
- Charged side chains. Kozlowski lists the ionizable side chains of seven amino acids: glutamate, aspartate, cysteine, tyrosine, histidine, lysine and arginine [4].
- The termini. The charge of the amine and carboxyl terminal groups also contributes to pI, and can greatly affect the pI of short peptides [4]. See N-terminus vs C-terminus for how the two ends are defined.
- The buffer. Net charge is strongly related to buffer pH and can be approximated with the Henderson–Hasselbalch equation [4].
- Proteins scale up the same idea. Lysozyme, rich in basic amino acids, has a high pI (11.0). Pepsin, rich in acidic amino acids, has a pI of about 1.0 [2].
Why peptide pI calculators give different answers
A theoretical pI is a calculation, and the answer depends on the pKa set behind it:
- pKa values are empirical. Kozlowski notes that the dissociation constants used are usually derived empirically and can vary substantially with experimental conditions such as temperature or buffer ionic strength [4].
- Many sets exist. His IPC benchmark compared IPC with 15 other pKa sets. On the peptide test set the RMSD was 0.251 pH units for IPC_peptide, 0.255 for Solomons, 0.262 for Lehninger, 0.325 for EMBOSS and 0.669 for Bjellqvist [4].
- Peptides are easier than proteins. IPC's average error was 0.25 pH units for peptides and 0.87 for proteins. Peptides are short and usually carry only a handful of charged residues [4].
- ExPASy's Compute pI/Mw uses the Bjellqvist pK values, which were defined from polypeptide migration between pH 4.5 and 7.3 in an immobilized pH gradient gel with 9.2 M and 9.8 M urea at 15 °C or 25 °C [6]. ProtParam's theoretical pI is calculated the same way [7].
- Known limits. ExPASy says predictions for highly basic proteins are yet to be studied, poor buffer capacity leads to greater error, and the program does not account for post-translational modifications [6].
Where pI matters in the lab
- Solubility. OpenStax notes that water solubility is usually lowest at the isoelectric point, where there is no net charge, and higher above and below it [2].
- Choosing a solvent. Bachem says overall acidic peptides can normally be reconstituted in basic solvents and overall basic peptides in acidic solvents. Before choosing a solvent, it says to count the acidic, basic and neutral residues, and it adds that the nature of the N- and C-terminal functionality has to be taken into consideration [8]. Bachem also warns that free cysteines oxidize rapidly to disulfides at pH above 7 [8]; see what a disulfide bond is.
- Electrophoresis. In a buffer of a given pH, molecules above their pI are negatively charged and migrate toward the positive electrode, and those below their pI migrate toward the negative electrode [2].
- Isoelectric focusing. Bjellqvist and colleagues measured the focusing positions of 29 polypeptides in narrow-range immobilized pH gradients. The pI values calculated from their sequences matched the experimental values with good accuracy [5]. Kozlowski also describes proteomics workflows in which a sample is digested by trypsin into short peptides and fractionated by isoelectric focusing before mass spectrometry [4].
Frequently Asked Questions
What is the isoelectric point?
The pH at which an amino acid or peptide carries, on average, no net charge [3].
How do you find the pI of an amino acid?
Average the two pKa values that involve the neutral zwitterion: pKa1 and pKa2 for neutral side chains, the two lowest for acidic side chains, the two highest for basic side chains [2].
What is the pI of glycine?
5.97 in the OpenStax table [1].
Is a peptide positive or negative below its pI?
Positive. Below the pI it is mainly protonated; above the pI it is mainly deprotonated and negative [2].
Why is solubility lowest at the pI?
At the pI there is no net charge. OpenStax notes solubility is usually lowest there and higher on either side [2].
Why do pI calculators disagree?
They use different pKa sets, and pKa values shift with conditions such as temperature and ionic strength [4].
Bottom line
The isoelectric point is the pH of zero net charge [3]. For a single amino acid it is the average of two pKa values [2]. For a peptide it reflects the termini plus every charged side chain [4], and it shapes solubility [2][8] and how the molecule moves in electrophoresis and isoelectric focusing [2][5]. Any calculated pI is an estimate tied to its pKa set [4][6].
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