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): N-terminus 9.0, C-terminus 3.1; Asp 3.9, Glu 4.1, His 6.0, Cys 8.3, Tyr 10.5, Lys 10.5, Arg 12.5. Different textbooks use slightly different values — yours may vary by a few tenths.