Isoelectric Point Calculator

The isoelectric point (pI) is the pH at which an amino acid or protein carries no net electrical charge, meaning the positive charges from protonated groups exactly balance the negative charges from deprotonated groups. At this pH the molecule does not migrate in an electric field.

Average the two pKa values that flank the neutral form of the molecule. For an amino acid with no ionizable side chain, this is simply pI = (pKa1 + pKa2) / 2, the average of the alpha-carboxyl and alpha-amino group pKa values.

Aspartate and glutamate have three ionizable groups instead of two: the alpha-carboxyl, the alpha-amino, and an extra side-chain carboxyl. As pH rises from very acidic, the alpha-carboxyl deprotonates first, then the side-chain carboxyl, then the alpha-amino. The neutral form sits between the first two, so pI = (pKa1 + pKa(R)) / 2.

Lysine, arginine, and histidine have an extra basic side-chain group (an amine, guanidinium, or imidazole). The neutral form sits between the alpha-amino group's pKa and the side chain's pKa, so pI = (pKa2 + pKa(R)) / 2.

It sums the fractional charge of every ionizable group in the molecule as a continuous function of pH, using the Henderson-Hasselbalch equation for each group. The pI is the pH where that summed net charge crosses zero. For a well-separated pair of flanking pKa values, this crossing point matches the simple averaging formula almost exactly, which is what this calculator's chart demonstrates.

No. pKa describes a single ionizable group's own acid dissociation constant. pI describes the pH at which the whole molecule, which may have two or more ionizable groups, has zero net charge. pI is calculated from two of the molecule's pKa values, but it is not itself a pKa.

At its pI, a protein has minimum net charge and minimum charge-based solubility in water, since there is no electrostatic repulsion between molecules to keep them apart in solution. This is exploited in isoelectric focusing (a protein purification and analysis technique) and explains why proteins tend to precipitate near their pI, a fact used directly by the salting-out relationship in the Protein Solubility Calculator.

Glycine, the simplest amino acid with no ionizable side chain, has an alpha-carboxyl pKa1 of about 2.34 and an alpha-amino pKa2 of about 9.60, giving a pI of about 5.97.

Lysine has an alpha-carboxyl pKa1 of about 2.18, an alpha-amino pKa2 of about 8.95, and a basic side-chain (epsilon-amino) pKa(R) of about 10.53. Its pI averages pKa2 and pKa(R), giving about 9.74, well above neutral pH because lysine is a basic amino acid.

This calculator computes pI for a single amino acid (or any molecule described by two or three known pKa values), the same building block used in more advanced protein pI predictions. A full peptide or protein pI calculation sums contributions from every ionizable residue in the sequence plus the free N-terminus and C-terminus, which is a larger charge-balance problem built on the same Henderson-Hasselbalch principle shown in this calculator's chart.

Yes, slightly. pKa values themselves shift a small amount with temperature and ionic strength, so a molecule's measured pI can shift by a few tenths of a pH unit under different experimental conditions. This calculator uses whichever pKa values you enter, so it reflects whatever conditions those pKa values were measured under.