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Isoelectric Point Calculator

Chemistry

Calculate the isoelectric point (pI) and net charge of a protein or amino acid at any pH. Uses Henderson-Hasselbalch equation with standard pKa values.

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Isoelectric Point (pI)

6.06
Net Charge at pH
-0.002
pKa (α-NH₃⁺)
9.78
pKa (α-COOH)
2.35

This calculator computes your Isoelectric Point (pI), Net Charge at pH, pKa (α-NH₃⁺), pKa (α-COOH) from the values you enter.

Inputs
Amino Acid / ResidueSolution pH
Outputs
Isoelectric Point (pI)Net Charge at pHpKa (α-NH₃⁺)pKa (α-COOH)

What is a Isoelectric Point?

The Isoelectric Point Calculator returns the isoelectric point (pI) and net charge at a given pH for all 20 standard amino acids. Select the amino acid and enter a pH to see the net charge computed using the Henderson-Hasselbalch equation applied to each ionisable group.

The isoelectric point is the pH at which a molecule carries zero net electrical charge. For single amino acids, pI is the average of the two pKa values flanking the neutral (zwitterionic) form. Net charge at any pH is computed by summing the fractional charges on each ionisable group (α-COOH, α-NH₃⁺, and side chain if present) from the Henderson-Hasselbalch equation. This determines whether the molecule will migrate toward the anode (negative charge, above pI) or cathode (positive charge, below pI) in an electric field — fundamental to electrophoretic separation.

For buffer calculations using amino acids as pH buffers (e.g., glycine buffer pH 2.3–3.6 or 8.6–10.0), the Henderson-Hasselbalch Calculator computes buffer pH from pKa and component ratios. For enzyme characterisation, the Michaelis-Menten Calculator and Enzyme Activity Calculator analyse catalytic behaviour.

How to use this Isoelectric Point calculator

  1. Select the amino acid from the dropdown (e.g., Glycine for simplest case; Aspartic acid for acidic pI; Lysine for basic pI).
  2. Enter the Solution pH — use the slider to scan across pH values.
  3. Read pI — the isoelectric point from standard reference data (pKa values from IUPAC reference tables).
  4. Read Net Charge at pH — positive value = cationic (below pI); negative = anionic (above pI); ~0 = at/near pI.
  5. For ion exchange: if you need amino acid/protein to bind DEAE anion exchanger (binds anions), set pH > pI; for CM cation exchanger (binds cations), set pH < pI.

Formula & Methodology

Net charge calculation (Henderson-Hasselbalch):

For each ionisable group:   Acidic group (COOH, side-chain acid):     charge = −1 / (1 + 10^(pKa − pH))    Basic group (NH₃⁺, side-chain amine):     charge = +1 / (1 + 10^(pH − pKa))  Net charge = Σ (charge contributions from all groups) pI = pH where net charge = 0

pI calculation for simple amino acids:

No ionisable side chain (Gly, Ala, Val, ...):   pI = (pKa_COOH + pKa_NH3) / 2  Acidic side chain (Asp, Glu):   pI = (pKa_COOH + pKa_sidechain) / 2   [neutral species: zwitterion but sidechain protonated]  Basic side chain (Lys, Arg, His):   pI = (pKa_NH3 + pKa_sidechain) / 2   [neutral species: zwitterion but sidechain protonated]

Worked example — Lysine (Lys, K):

pKa values: α-COOH = 2.16; α-NH₃⁺ = 9.06; ε-NH₃⁺ (side chain) = 10.54.

pI = (pKa_alpha_NH3 + pKa_side_chain) / 2 = (9.06 + 10.54) / 2 = 9.80

At pH 7 (physiological): α-COOH fully deprotonated (−1); α-NH₃⁺ mostly protonated (+1, pKa 9.06 >> 7); ε-NH₃⁺ fully protonated (+1, pKa 10.54 >> 7). Net charge ≈ +1. Lysine is cationic at physiological pH — which is why it binds negatively charged DNA and RNA. Histones (the protein spools around which DNA wraps in chromosomes) are extremely Lys-rich (pI ~10.8), maintaining tight electrostatic interaction with negatively charged DNA phosphates at cellular pH 7.2. This principle is taught in every Indian university biochemistry course and is a frequent NEET PG and MD entrance exam question.

Frequently Asked Questions

The isoelectric point (pI) is the pH at which a molecule carries zero net electrical charge. At pH = pI, the positive charges on protonated groups (α-NH₃⁺, Lys, Arg, His side chains) exactly balance the negative charges on deprotonated groups (α-COO⁻, Asp, Glu side chains, Tyr, Cys). Below pI: the molecule is positively charged overall (net positive). Above pI: negatively charged (net negative). At pI, solubility is minimised (reduced electrostatic repulsion between molecules) and electrophoretic mobility is zero — the basis of isoelectric focusing (IEF) separation.
For amino acids with only two ionisable groups (non-polar amino acids: Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, Trp): pI = (pKa1 + pKa2)/2, where pKa1 = α-COOH and pKa2 = α-NH₃⁺. Example: Glycine pKa1=2.35, pKa2=9.78 → pI = (2.35+9.78)/2 = 6.065. For amino acids with an ionisable side chain: pI = average of the two pKa values that flank the neutral species: acidic amino acids (Asp pI ≈ 2.98): pI = (pKa_COOH + pKa_sidechain)/2; basic amino acids (Lys pI ≈ 9.60): pI = (pKa_amino + pKa_sidechain)/2.
Select the amino acid from the dropdown (all 20 standard amino acids). Enter a pH value (0–14) to see the net charge at that pH. The calculator returns pI (the isoelectric point from standard reference tables), net charge at the entered pH (using Henderson-Hasselbalch equation for each ionisable group), and the pKa values for the alpha-amino and alpha-carboxyl groups. At pH = pI, net charge ≈ 0.
pI is used in: (1) Isoelectric focusing (IEF): proteins are separated in a pH gradient — each protein migrates to its pI and stops (zero mobility). Used in 2D gel electrophoresis (2D-PAGE) for proteomics, which is standard in AIIMS, NCBS, and IISc research labs. (2) Ion exchange chromatography: at pH < pI, protein is positive → binds cation exchangers (e.g., CM-Sepharose). At pH > pI, protein is negative → binds anion exchangers (e.g., DEAE-Sepharose). (3) Protein crystallisation: crystals most easily form near pI (minimum solubility). (4) Enzyme stability: most enzymes show maximum stability near their pI.
Common protein pI values: Pepsin (gastric): pI ≈ 1.0 (very acidic, active at gastric pH ~2). Casein (milk protein): pI ≈ 4.6 — milk proteins precipitate at pH 4.6 (basis of paneer/curd making in India — lactic acid fermentation drops pH to pI). Human albumin (BSA): pI ≈ 4.7. Myoglobin: pI ≈ 7.0. Haemoglobin: pI ≈ 6.8. Lysozyme (egg white): pI ≈ 10.7 (very basic, high Lys/Arg content). Histones: pI ≈ 10.8–11.3 (rich in Lys and Arg for DNA binding). Cytochrome c: pI ≈ 10.0. The very basic pI of histones enables electrostatic binding to negatively charged DNA at physiological pH.
At pH = pI, proteins are least soluble — intermolecular electrostatic repulsion is minimised, and proteins aggregate and precipitate. This is the basis of isoelectric precipitation: (1) Casein precipitation at pH 4.6 in milk acidification (Indian paneer, dhahi/curd preparation). (2) Acid precipitation of soy protein isolate at pH 4.5 in soy protein processing (major Indian edible protein industry). (3) Egg white coagulation at pH adjustment. For industrial protein recovery, precipitating at pI removes bulk protein, which is then resolubilised and purified — a standard unit operation described in Perry's Chemical Engineers' Handbook and practised at AMUL's protein processing facilities.
A zwitterion ('double ion') has both a positive and negative charge simultaneously with zero net charge. All amino acids exist predominantly as zwitterions at their pI: the α-COOH is deprotonated (-COO⁻) and the α-NH₂ is protonated (-NH₃⁺) simultaneously. The neutral 'uncharged' form (both groups at their non-ionised state: COOH and NH₂) is actually very rare under aqueous conditions — the zwitterion is thermodynamically favoured by ~3–5 kcal/mol due to dipole stabilisation by water. This is why amino acids have high melting points (~200°C), are non-volatile, and have crystal structures — properties of ionic compounds, not covalent molecules.
Human insulin pI ≈ 5.4. At physiological blood pH 7.4, insulin is negatively charged — this affects its distribution and receptor binding. Insulin formulations manipulate pH for stability: (1) Regular insulin: dissolved at pH 7.4 (soluble). (2) NPH (neutral protamine Hagedorn) insulin: complexed with protamine (a basic protein, high pI ~11) and zinc at pH 7.4 — forms a suspension (slow absorption). (3) Glargine (Lantus): formulated at pH 4 (below insulin pI) → precipitates at subcutaneous pH 7.4 → slow release. India is the world's diabetes capital with ~77 million diabetics — Biocon (Bangalore) manufactures biosimilar insulin, requiring precise pH/pI-based formulation to match Eli Lilly's reference products for CDSCO approval.
Henderson-Hasselbalch: pH = pKa + log([A⁻]/[HA]), where [A⁻]/[HA] = ratio of deprotonated to protonated form. Rearranged for fraction deprotonated: f = 1/(1 + 10^(pKa - pH)). For each ionisable group: at pH >> pKa → fully deprotonated; at pH << pKa → fully protonated. Net charge = Σ(charges × fractions) for all ionisable groups. pI is the pH where net charge = 0 — found by iterating pH until net charge crosses zero. The [Henderson-Hasselbalch Calculator](/henderson-hasselbalch-calculator/) applies this equation to buffer systems (weak acid + conjugate base), which uses the same underlying equilibrium principles.
Yes — in SDS-PAGE, proteins are coated with SDS (negatively charged), so they migrate by size regardless of pI. But in native PAGE and isoelectric focusing (IEF), charge matters. In IEF: protein in pH gradient migrates until reaching its pI (no charge → no movement). 2D-PAGE = IEF (by pI) in first dimension + SDS-PAGE (by MW) in second dimension — separates complex mixtures of thousands of proteins. The IPG strips (immobilised pH gradient) used in Indian proteomics labs (AIIMS, CCMB, TIFR) are specified by pI range (pH 3–10, 4–7, 6–11), and researchers choose the strip based on expected pI of their proteins of interest.