Protein Net Charge Calculator (pH-Dependent)

Henderson-Hasselbalch Equation and Protein Net Charge

Proteins carry several types of ionizable groups: the N-terminal α-amino group (basic), the C-terminal α-carboxyl group (acidic), and seven side-chain types — Asp, Glu, His, Cys, Tyr, Lys, and Arg. At a given pH, the charged fraction of each group is determined by the Henderson-Hasselbalch equation.

Basic groups (N-terminus, His, Lys, Arg; carry +1 charge when protonated):

f(+) = 1 / (1 + 10^(pH − pKa))

Acidic groups (C-terminus, Asp, Glu, Cys, Tyr; carry −1 charge when deprotonated):

f(−) = 10^(pH − pKa) / (1 + 10^(pH − pKa))

If a group has n residues, its net charge contribution is +n·f(+) (basic) or −n·f(−) (acidic). Summing over all groups gives the theoretical net charge:

Z(pH) = Σ(basic: +n · f(+)) + Σ(acidic: −n · f(−))

The isoelectric point (pI) is the pH at which Z = 0; this tool finds it automatically by bisection over pH 0–14.

pKa Reference Values for Each Group

This tool uses the pKa values from EMBOSS iep, the same set used by the Protein Molecular Weight / Isoelectric Point Calculator on this site — two tools on the same site giving different numbers for the same groups would leave the reader unsure which to trust (before alignment, the two tools returned pI values of 3.40 and 3.61 for insulin A chain). Source: EMBOSS iep documentation.

Note: Arg’s pKa (12.5) is well above the physiological pH range; at pH 4–12, it effectively carries +1 charge at all times (f(+) > 0.9999).

Different pKa scales differ substantially and will directly change the result — this must be stated clearly. ExPASy’s Compute pI/Mw uses an empirical scale derived by Bjellqvist et al. from the migration positions of proteins in immobilized pH gradient gels (Bjellqvist B, Hughes GJ, Pasquali C et al., Electrophoresis 1993;14:1023-31, PMID: 8125050); its side-chain values differ item by item from the table above (e.g. His ≈ 5.98 vs. 6.5 here, Lys ≈ 10.0 vs. 10.8, Arg ≈ 12.0 vs. 12.5), and the N-terminus pKa varies with the identity of the first residue. The difference can be quantified directly: at pH 7.4, His with pKa 6.5 gives f(+) = 0.1118, while pKa 5.98 gives 0.0366 — a roughly 3-fold difference. Therefore — This tool’s values will not match ExPASy digit-for-digit, most noticeably for His-rich proteins; when comparing against a published pI, always confirm which pKa scale was used. This tool is designed for directional decisions (see Scope and Common Mistakes below), not as a precise reproduction of ExPASy.

Scope and Common Mistakes

Applicable situations: directional assessment of linear peptides and folded proteins — which IEX resin to choose and what sign of charge a protein carries at a given pH.

Not applicable, or requiring extra care: - Precise charge of folded proteins: His pKa in a hydrophobic core can be as low as 4; Cys adjacent to a positive charge can drop to 6; these shifts are structure-dependent and cannot be captured here. - Post-translational modifications: each phosphorylation site contributes an extra −1 to −2 charge at physiological pH; O-glycosylation has limited effect on net charge, but phosphorylation requires manual correction. - Disulfide bonds: paired Cys thiols no longer ionize; enter the correct number of disulfide bonds.

The most common mistake: deciding precipitation conditions from pI alone. Zero net charge (at the pI) is a necessary condition for the loss of electrostatic repulsion, but aggregation is driven by hydrophobic interactions, which are independent. IEX binding typically requires |Z| ≥ 2 and a working pH at least 1 unit away from the pI.

Worked Example (Verifiable by Hand)

Bradykinin (RPPGFSPFR, 9 amino acids)

The sequence contains only 2 Arg residues; there are no Asp, Glu, His, Cys, Tyr, or Lys, leaving only three terms. At pH = 7.4:

N-terminus (basic, pKa 8.6)   f(+) = 1 / (1 + 10^(7.4 − 8.6))  = +0.941
Arg × 2  (basic, pKa 12.5)    f(+) = 1 / (1 + 10^(7.4 − 12.5)) = +2.000
C-terminus (acidic, pKa 3.6)  f(−) = 10^(7.4 − 3.6) / (1 + 10^(7.4 − 3.6)) = −1.000

Z = +0.941 + 2.000 − 1.000 = +1.94 (these four values are what the tool will display)

Bradykinin pI ≈ 12.5 (dominated by the two Arg residues); at pH 7.4 its net charge is approximately +1.9, giving effective retention on a CEX column and no binding to AEX — consistent with reported chromatographic behavior.