The curve is net charge vs pH. The pI is where it crosses zero (dashed line). Your selected pH is the vertical marker.
Step-by-step: every ionizable group
Each group's charge comes from Henderson-Hasselbalch. Bases (N-terminus, His, Lys, Arg) are positive when protonated; acids (C-terminus, Asp, Glu, Cys, Tyr) are negative when deprotonated.
| Group | Type | pKa | Charge at pH 7.0 |
|---|
How to calculate the pI of a peptide by hand
List every ionizable group (the N-terminus, the C-terminus, and any charged side chains). At a given pH, each positive group contributes +1/(1+10^(pH−pKa)) and each negative group contributes −1/(1+10^(pKa−pH)). The net charge is the sum. The pI is the pH where that sum equals zero. You can find it by averaging the two pKa values that bracket the neutral species. This tool does all of that live so you can check your hand calculations.
pKa values used (standard textbook set, matching the amino acid titration curve tool's side-chain values): N-terminus 9.0, C-terminus 3.1; Asp 3.65, Glu 4.25, His 6.0, Cys 8.18, Tyr 10.07, Lys 10.53, Arg 12.48. Different textbooks use slightly different values, yours may vary by a few tenths.
Want the full by-hand method with worked examples? See How to calculate pI without a calculator.
Related tools: Henderson-Hasselbalch buffer calculator · Amino acid titration curve · Protein MW & extinction coefficient · Amino acid chart (all 20) · all biochem tools.
Worked example: pI of DKHEYR by hand
DKHEYR has 8 ionizable groups: the N-terminus, C-terminus, and one charged side chain per residue. At pH 7.0, Henderson-Hasselbalch gives each group's fractional charge:
| Group | pKa | Type | Charge at pH 7.0 |
|---|---|---|---|
| N-terminus | 9.0 | base | +0.99 |
| Asp (D) | 3.65 | acid | −1.00 |
| Lys (K) | 10.53 | base | +1.00 |
| His (H) | 6.0 | base | +0.09 |
| Glu (E) | 4.25 | acid | −1.00 |
| Tyr (Y) | 10.07 | acid | 0.00 |
| Arg (R) | 12.48 | base | +1.00 |
| C-terminus | 3.1 | acid | −1.00 |
| Net charge at pH 7.0 | +0.08 | ||
The three acidic groups (D, E, C-terminus) are almost fully deprotonated (each ≈ −1), and three of the four basic groups (N-terminus, K, R) are almost fully protonated (each ≈ +1), those roughly cancel. Tyrosine's phenol is acidic too, but because its pKa of 10.07 is far above 7, at pH 7 it's still protonated and essentially neutral (≈ 0), an acid only sheds its proton once the pH climbs past its pKa. The one group still transitioning at pH 7 is His (pKa 6.0, so it's only about 9% protonated, ≈ +0.09), which is why the net charge is a small positive number (+0.08) rather than exactly zero.
Since the net charge is slightly positive at pH 7 and charge decreases as pH rises (every group loses a proton), the pI must sit a bit above 7. Solving where the sum crosses zero gives pI ≈ 7.48, matching what the calculator above shows if you load DKHEYR and slide to pH 7.48.
FAQ
What is the isoelectric point (pI) of a peptide?
The pH at which the net charge is exactly zero. Below the pI the peptide carries net positive charge; above it, net negative.
How do you calculate pI without a calculator?
For a peptide with only two ionizable groups controlling the neutral region, average their two pKa values. For anything more complex (like DKHEYR above), sum each group's charge at a trial pH and adjust until the sum hits zero, the general method this tool automates.
Does this same approach work for whole proteins?
Yes in principle, sum every ionizable group and find the zero crossing. In practice a folded protein's local environment shifts individual pKa values away from the free-amino-acid values used here, so a calculated protein pI is an approximation; measured pI (isoelectric focusing) can differ by up to roughly half a pH unit.
Why doesn't my calculated pI match a textbook answer?
Different textbooks and software use slightly different pKa reference sets for the same side chains, which shifts the calculated pI by a few tenths of a pH unit. Check which pKa table your problem set expects.
Practice problems
1. What is the net charge of the dipeptide KR (Lys-Arg) at pH 7.0?
Show answer
2. What is the isoelectric point (pI) of the dipeptide DD (Asp-Asp)?
Show answer
Sources and how to cite this page
The pKa values are the standard set tabulated in Lehninger Principles of Biochemistry (Nelson & Cox). Net charge at a given pH is computed by summing the fractional charge on every ionizable group, each from the Henderson-Hasselbalch relation, including the free N-terminal α-amino group, the free C-terminal α-carboxyl group, and the seven ionizable side chains (Asp, Glu, His, Cys, Tyr, Lys, Arg). The pI is found by solving numerically for the pH where that sum crosses zero.
What this does not model. These are free-amino-acid pKa values. In a folded protein, nearby charges, hydrogen bonding, and the local dielectric can shift a side-chain pKa by more than a full unit, so a calculated pI is a good estimate for a short peptide and only an approximation for a folded protein. Measured pI from isoelectric focusing is the ground truth.
Why published pKa values differ slightly. pKa is measured, not derived, so a tabulated value depends on temperature and ionic strength. Values here are for dilute aqueous solution near 25 °C. Differences of roughly 0.1 to 0.3 pKa units between references are normal and do not mean one is wrong. Cite the source you were taught from and stay consistent with it.