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Protein Isoelectric Point (pI) Calculator

Estimate the isoelectric point of a protein or peptide from its sequence with a choice of pKa set, and plot the net charge against pH, with the charge at a pH you choose.

Formula

Z=∑+11+10 pH−pKa−∑−11+10 pKa−pHZ = \sum_{+} \dfrac{1}{1 + 10^{\,\mathrm{pH}-\mathrm{p}K_{\mathrm a}}} - \sum_{-} \dfrac{1}{1 + 10^{\,\mathrm{p}K_{\mathrm a}-\mathrm{pH}}}
pI: the pH at which Z=0\mathrm{p}I:\ \text{the pH at which } Z = 0
pKa\mathrm{p}K_{\mathrm{a}}
the pH at which half of a group is ionised
positive groups\text{positive groups}
the N terminus, Lys, Arg and His
negative groups\text{negative groups}
the C terminus, Asp, Glu, Cys and Tyr

How it works

Each ionisable group contributes a charge that depends on the pH and its pKa, through the Henderson-Hasselbalch relationship. Adding the contributions of every group gives the net charge at a pH, which falls as the pH rises. The isoelectric point is where it crosses zero, and is found here by bisection.

The pKa values of the groups are not fixed. They shift with the neighbouring residues and with folding, so any calculated pI is an estimate. Two common sets of values are offered, the Lehninger textbook values and those used by the ExPASy tool, and they can give answers differing by a pH unit or more for the same sequence, which is shown in the example. A sequence-based pI is a useful guide for choosing a buffer, an ion-exchange resin or an isoelectric-focusing range, and should be checked by experiment.

Worked example

Angiotensin II, DRVYIHPF, with both sets of pKa values, and the charge at pH 7.4.

  1. Lehninger values: the net charge crosses zero at pH 7.77.
  2. ExPASy values: it crosses zero at pH 6.74.
  3. At pH 7.4 (Lehninger) the net charge is +0.03.

pI 7.77 (Lehninger) or 6.74 (ExPASy) for the same peptide: a difference of more than a pH unit from the choice of pKa alone.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Every ionisable group behaves independently with the pKa listed, with no interaction between charges.
  • Free termini, and no modifications such as phosphate or acetyl groups.
  • The ExPASy set is used without that tool's residue-specific terminal corrections.

Common mistakes

  • Treating a calculated pI as exact. A measured pI from isoelectric focusing often differs by a unit or more.
  • Forgetting that the protein is least soluble near its pI, and that a buffer close to it can cause precipitation.
  • Using the whole-precursor sequence for a mature protein with a cleaved signal peptide.

Related tools

Protein and Peptide Molecular Weight CalculatorAverage or monoisotopic mass from a sequence, with end groupsOpen calculatorAmino Acid Composition CalculatorResidue counts, GRAVY, aliphatic index and charge contentOpen calculatorHenderson-Hasselbalch CalculatorBuffer pH, base-to-acid ratio, or pKaOpen calculatorProtein Concentration Calculator (A280, A205)Concentration from absorbance, and mg/mL ⇄ µMOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.