The isoelectric point, pI, is the pH at which a peptide’s net charge is zero. It is not a fixed property looked up on a table; it is the answer to an equation, and this post explains how that equation is set up and solved.
Net charge is a sum, not a lookup
A peptide’s net charge at a given pH is the sum of the charge contributed by every ionizable group in it: the free N-terminal amine, the free C-terminal carboxyl, and the side chains of residues that can gain or lose a proton (aspartate, glutamate, cysteine, tyrosine, histidine, lysine, arginine). Each group’s contribution depends on how far the surrounding pH is from that group’s own pKa, following the Henderson-Hasselbalch relationship. A basic group (like the N-terminus or lysine’s side chain) contributes close to +1 at low pH and close to 0 at high pH; an acidic group (like the C-terminus or aspartate) contributes close to 0 at low pH and close to -1 at high pH.
This site’s net charge calculator sums exactly that: one term per ionizable group, evaluated at whatever pH you enter. The Henderson-Hasselbalch and net charge post walks through that sum term by term.
What is the isoelectric point (pI) of a peptide?
Net charge is a smoothly decreasing function of pH: at very low pH every ionizable group is protonated and the peptide carries its maximum positive charge, and at very high pH every group is deprotonated and it carries its maximum negative charge. Somewhere in between, the positive and negative contributions cancel exactly. That crossing point is the isoelectric point.
There is no closed-form formula that solves for that pH directly when several ionizable groups with different pKa values are involved, so it has to be found numerically.
Bisection: narrowing the interval
The isoelectric point calculator finds it with a bisection search. Bisection works on any function that changes sign across an interval: start with a low bound where the function is known to be positive and a high bound where it is known to be negative (here, pH 0 and pH 14 bracket every realistic net charge crossing), evaluate the function at the midpoint, and replace whichever bound has the same sign as the midpoint with the midpoint itself. The bracket shrinks by half on every step and pI is guaranteed to stay inside it.
This calculator’s implementation caps the search at 100 iterations and stops early once the bracket narrows to within 1e-6 of a pH unit, which converges to a stable four-decimal result well within that iteration cap for any real sequence. Each step is the same net-charge sum used by the net charge calculator, evaluated at a new candidate pH, so the pI calculation is not a separate method: it is the net charge equation, solved rather than evaluated.
The pKa scale behind the numbers
Every pKa value used in this sum, for both the terminal groups and the ionizable side chains, comes from a single published scale: Bjellqvist et al. 1993, the same scale ExPASy’s Compute pI/Mw tool uses. It is one of several published side-chain pKa scales, not a universal physical constant; different scales (each measured or fitted under different conditions) give slightly different pI values for the same sequence. This site fixes one scale throughout so results stay internally consistent and auditable against a named source, rather than offering a menu of scales with no way to know which one a given output used.
Seeing the whole curve at once
A single pI value is one point on a larger picture: net charge across the full pH range. The charge vs. pH curve calculator plots that curve, sampling net charge at points from pH 0 to pH 14, so you can see how steeply charge changes near the pI and how flat it is far away from it, rather than only the single crossing point.
A related, simpler count
pI answers “at what pH is charge zero”; a simpler related question is “what fraction of the residues in this sequence are charged at all”, regardless of pH. The charged residue fraction calculator answers that second question by counting acidic and basic residues as a percentage of the sequence, which is a useful quick read on how much of a peptide’s identity is driven by charge before working through a full pI calculation.
What pI does not claim
The Henderson-Hasselbalch method used here treats each ionizable group as independent, with a fixed pKa regardless of the peptide’s three-dimensional structure or the presence of neighboring charged groups. Real peptides can have local electrostatic effects that shift an individual group’s effective pKa away from the scale value. pI computed this way is a standard, widely used approximation, the same one behind familiar tools like ExPASy, not an experimentally measured value for your specific sequence.
In short, a peptide’s isoelectric point is the pH at which the sum of every ionizable group’s charge equals zero, found by a bisection search on the Bjellqvist pKa scale rather than a closed-form equation.
Primary scientific literature and citations
- Bjellqvist, B., Hughes, G. J., Pasquali, Ch., Paquet, N., Ravier, F., Sanchez, J. Ch., Frutiger, S., & Hochstrasser, D. (1993). The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis, 14(1), 1023-1031. PubMed PMID: 8125050. DOI: 10.1002/elps.11501401163. Established the empirical polypeptide pKa scale implemented in ExPASy Compute pI/Mw tool.
- Bjellqvist, B., Basse, B., Olsen, E., & Celis, J. E. (1994). Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions. Electrophoresis, 15(1), 529-539. PubMed PMID: 8055880. Refined terminal and side chain pKa values for accurate 2D gel isoelectric focusing predictions.
- Nelson, D. L., & Cox, M. M. (2021). Lehninger Principles of Biochemistry (8th ed.). W. H. Freeman / Macmillan Learning. Chapter 3: Amino Acids, Peptides, and Proteins. Comprehensive reference for amino acid titration behavior and isoelectric points.
- Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server. In J. M. Walker (Ed.), The Proteomics Protocols Handbook, Humana Press, 571-607. Documentation of the computational bisection root-finding method for theoretical pI calculations.