Compute the isoelectric point (pI) of any molecule by defining custom ionizable groups with their pKa values and type (acidic/basic). Visualize the net charge vs. pH titration curve, understand the Henderson-Hasselbalch behavior, and get accurate pI for proteins, peptides, and ampholytes.
The isoelectric point (pI) is the pH at which a molecule carries no net electrical charge. For amino acids, peptides, and proteins, the pI is a critical physicochemical property that influences solubility, electrophoretic mobility, crystallization behavior, and interactions with other biomolecules. At the pI, the sum of positive charges exactly balances the sum of negative charges.
Henderson–Hasselbalch equation for each ionizable group:
For acidic group (HA ⇌ A⁻ + H⁺): αdeprotonated = 1 / (1 + 10(pKa - pH))
For basic group (BH⁺ ⇌ B + H⁺): αprotonated = 1 / (1 + 10(pH - pKa))
Net charge Q(pH) = Σ (acidic groups: -αdeprot) + Σ (basic groups: +αprot). The pI is found by solving Q(pH) = 0 using numerical root-finding (bisection method).
Our calculator uses high‑precision bisection (pH range 0–14, tolerance 1e-6) to locate the exact pI. Additionally, the titration curve is rendered dynamically, helping you see how net charge changes with pH and confirming the isoelectric point visually.
Each ionizable group is characterized by its intrinsic pKa value, which depends on the local chemical environment. For standard amino acid side chains, pKa values are well documented (e.g., Asp ~3.9, Glu ~4.1, Lys ~10.5, Arg ~12.5, His ~6.0). Our calculator lets you define any set of pKa values, making it suitable for modified residues, non‑natural amino acids, small ampholytes, and even complex buffer systems. The underlying algorithm applies the Henderson‑Hasselbalch formalism and assumes independent, non‑interacting groups – a standard approximation widely used in biochemistry.
For large proteins with strong charge–charge interactions, the independent pKa model provides a first approximation; consider using a Poisson–Boltzmann solver (e.g., PDB2PQR, DelPhi) for higher precision when electrostatic coupling is significant. Within this limitation, the tool gives accurate pI values for small peptides, individual amino acids, and many practical applications.
In SDS‑PAGE, proteins are denatured and coated with negative charge, but native PAGE and isoelectric focusing (IEF) rely on intrinsic pI. A protein's pI determines its migration direction in a pH gradient. For example, human serum albumin (pI ≈ 4.7) migrates toward the anode at pH 7, while lysozyme (pI ≈ 11) moves toward the cathode. Our tool allows rapid pI prediction, aiding experimental design.
| Group | pKa (approx) | Type |
|---|---|---|
| α-COOH (C-terminal) | 2.0 – 2.4 | Acidic |
| α-NH₃⁺ (N-terminal) | 8.0 – 9.0 | Basic |
| Aspartic acid (side chain) | 3.9 | Acidic |
| Glutamic acid (side chain) | 4.1 | Acidic |
| Histidine (imidazole) | 6.0 | Basic |
| Cysteine (thiol) | 8.3 | Acidic |
| Tyrosine (phenol) | 10.1 | Acidic |
| Lysine (ε-NH₃⁺) | 10.5 | Basic |
| Arginine (guanidino) | 12.5 | Basic |
Because the net charge function Q(pH) is monotonic decreasing for ampholytes with multiple groups (under standard conditions), the bisection method guarantees convergence. Starting from pH = 0 (highly positive net charge) to pH = 14 (highly negative net charge), we iteratively narrow the interval until Q(pH) is within 1e-8 of zero. The algorithm handles any number of acidic and basic groups, making it suitable for complex molecules. For molecules with an even number of groups, the pI is the average of the two pKa values surrounding the zero‑charge region; our numeric method replicates that analytically.
Our implementation also computes the titration curve points (0.05 pH increments) for smooth canvas rendering, with optimized charge calculation per point.