Isoelectric point calculator
Estimate the pH at which a peptide carries no overall charge, and see how much the answer depends on the pKa values used.
pI: the pH where Σ charges = 0 (Henderson–Hasselbalch)
What is the isoelectric point?
The isoelectric point (pI) is the pH at which a peptide or protein carries no overall electric charge. Below its pI it is positively charged, above it negatively charged. At the pI a peptide often dissolves least, and it doesn't move in an electric field, which is how isoelectric focusing separates and measures it.
How the isoelectric point is calculated
Each ionisable group carries a fraction of a charge that depends on the pH and its pKa, from the Henderson–Hasselbalch equation. The groups are the free N- and C-termini and the side chains of Asp, Glu, His, Cys, Tyr, Lys and Arg. Adding them up gives the net charge, and the pI is the pH where that sum is zero. Cysteines in disulfide bridges, and termini that are acetylated or amidated, carry no charge.
Why the pKa sets give different answers
There is no single correct set of pKa values: a group's pKa shifts with its neighbours, the solvent, temperature and folding. Textbook sets come from free amino acids. Grimsley et al. (2009) averaged values measured in folded proteins, and Kozlowski (2016) fitted the IPC peptide set to measured peptide pIs, where it was the most accurate of the sets he compared. For a short peptide the estimates usually agree within a few tenths of a unit. A wide spread means the pI is uncertain and worth measuring. The pKa sets are as tabulated by Kozlowski (2016), Biology Direct 11:55.
The guide to isoelectric point and net charge works through the calculation by hand, and the peptide sequence properties tool plots charge across the whole pH range.