Isoelectric Point (pI) Calculator
Calculate the isoelectric point of any protein or peptide from its amino acid sequence — view the charge vs pH curve, compute net charge at physiological pH 7.4, and use the MCAT pI formula with full step-by-step working.
| Group | pKa | Charge type | Count | Charge at pI |
|---|
Select any amino acid to see its pKa values, which pI formula applies, and a full Henderson-Hasselbalch breakdown at any pH.
Calculate the exact net charge of a protein or peptide at any pH. Useful for predicting electrophoresis migration, ion exchange behavior, and solubility.
What Is the Isoelectric Point? — Definition and Meaning
This isoelectric point calculator computes the pI of any protein or peptide from its amino acid sequence, shows the full charge vs pH curve, and calculates net charge at physiological pH. The isoelectric point (pI, also written as pI or PI) is the pH at which a protein, peptide, or amino acid carries zero net charge — the number of positive charges exactly equals the number of negative charges.
The isoelectric pH is a fundamental property of every protein determined by its amino acid composition. It is not fixed by external conditions — only the amino acid sequence determines the isoelectric point. Understanding what an isoelectric point means is essential for electrophoresis, protein purification, and drug design.
What Does an Isoelectric Point of 9 Mean?
A protein with pI = 9 is a basic protein. At physiological pH 7.4, it carries a net positive charge because the solution pH (7.4) is below the protein's pI (9). Basic proteins like lysozyme (pI 11.35) and histones (pI > 10) are positively charged at pH 7.4 and bind negatively charged molecules like DNA and phospholipids.
Practical Importance of the Isoelectric Point
- Minimum solubility at pI: Proteins aggregate and precipitate at their isoelectric point because there is no electrostatic repulsion between molecules. This is why milk curdles when acidified — casein (pI ≈ 4.6) precipitates at its isoelectric point.
- Isoelectric focusing (IEF): Separates proteins by their isoelectric point in a pH gradient gel — each protein migrates until it reaches its pI zone and stops.
- Ion exchange chromatography: At pH below pI, protein binds cation exchange resins; at pH above pI, it binds anion exchange resins.
- Electrophoresis: At pH > pI, protein migrates toward the positive electrode (anode); at pH < pI, it migrates toward the cathode.
How to Calculate the Isoelectric Point — Step-by-Step
There are two methods to calculate isoelectric point depending on whether you are working with a single amino acid or a peptide/protein with multiple ionizable groups.
Method 1 — Simple Formula for Single Amino Acids (MCAT Method)
For a single free amino acid, the isoelectric point formula is the average of two pKa values. Which two depends on the type of amino acid:
Worked Example — Alanine (neutral amino acid)
- Identify: Alanine is a neutral amino acid (non-ionizable side chain)
- pKa1 (alpha-carboxyl) = 2.35; pKa2 (alpha-amino) = 9.69
- Apply neutral formula: pI = (2.35 + 9.69) / 2 = 6.02
- Interpretation: Alanine is slightly acidic compared to pure neutral (pH 7)
Worked Example — Aspartate (acidic amino acid)
- Identify: Aspartate is acidic (has an ionizable carboxyl side chain)
- pKa1 (alpha-carboxyl) = 1.99; pKa2 (alpha-amino) = 9.90; pKa3 (side chain -COOH) = 3.90
- For acidic AA: pI = (pKa1 + pKa3) / 2 = (1.99 + 3.90) / 2 = 2.95
- Why? The isoelectric point falls between the two acidic groups — Asp is zwitterionic only when both acidic groups are around their pKa
Worked Example — Lysine (basic amino acid)
- Identify: Lysine is basic (has an ionizable amino side chain)
- pKa1 = 2.16; pKa2 (alpha-amino) = 9.06; pKa3 (side chain -NH₂) = 10.54
- For basic AA: pI = (pKa2 + pKa3) / 2 = (9.06 + 10.54) / 2 = 9.80
Method 2 — Iterative Charge-Balance for Peptides and Proteins
For peptides and proteins with multiple ionizable groups, the simple formula fails. The correct method uses the Henderson-Hasselbalch equation for each ionizable group and finds the pH where the sum of all charges = 0.
- Identify all ionizable groups: N-terminus (pKa 8.0), C-terminus (pKa 3.1), and any ionizable side chains: Asp(D) pKa 3.65, Glu(E) pKa 4.25, His(H) pKa 6.00, Cys(C) pKa 8.18, Tyr(Y) pKa 10.07, Lys(K) pKa 10.53, Arg(R) pKa 12.48
- Write net charge equation: For each positive group (N-term, R, K, H): charge contribution = +n/(1 + 10^(pH − pKa)). For each negative group (C-term, D, E, C, Y): charge contribution = −n/(1 + 10^(pKa − pH))
- Bisection search: Start with lo=0, hi=14. At pH=mid, if net charge > 0, the pI is higher → lo=mid. If net charge < 0, the pI is lower → hi=mid. Repeat until convergence (<0.001)
Worked Example — Peptide ACDE (4 residues)
- Ionizable groups: N-term (pKa 8.0, +), C-term (pKa 3.1, −), C (pKa 8.18, −), D (pKa 3.65, −), E (pKa 4.25, −)
- At pH 3.0: charge ≈ +0.91 (N-term) − 0.29 (C-term) − 0.03 (C) − 0.0002 (D) − 0.00007 (E) ≈ +0.59 (positive)
- At pH 5.0: all acidic groups mostly deprotonated → charge ≈ −0.78 (negative)
- Bisection converges to pI ≈ 3.37 where net charge ≈ 0
Isoelectric Point Formula — The Simple (pKa1+pKa2)/2 Method
The isoelectric point formula pI = (pKa1 + pKa2) / 2 is the standard MCAT-tested approach. It works for single free amino acids only. The key is knowing which two pKa values to average:
| AA | Name | pKa₁ (-COOH) | pKa₂ (-NH₃) | pKa₃ (side chain) | pI | Type |
|---|---|---|---|---|---|---|
| A | Alanine | 2.35 | 9.69 | — | 6.02 | Neutral |
| R | Arginine | 1.83 | 8.99 | 12.48 | 10.76 | Basic |
| N | Asparagine | 2.14 | 8.72 | — | 5.41 | Neutral |
| D | Aspartate | 1.99 | 9.90 | 3.90 | 2.85 | Acidic |
| C | Cysteine | 1.92 | 10.70 | 8.18 | 5.07 | Special |
| E | Glutamate | 2.10 | 9.47 | 4.25 | 3.22 | Acidic |
| Q | Glutamine | 2.17 | 9.13 | — | 5.65 | Neutral |
| G | Glycine | 2.35 | 9.87 | — | 6.06 | Neutral |
| H | Histidine | 1.80 | 9.33 | 6.04 | 7.60 | Basic |
| I | Isoleucine | 2.32 | 9.76 | — | 6.04 | Neutral |
| L | Leucine | 2.33 | 9.74 | — | 6.04 | Neutral |
| K | Lysine | 2.16 | 9.06 | 10.54 | 9.60 | Basic |
| M | Methionine | 2.13 | 9.28 | — | 5.74 | Neutral |
| F | Phenylalanine | 2.20 | 9.31 | — | 5.91 | Neutral |
| P | Proline | 1.95 | 10.64 | — | 6.30 | Special |
| S | Serine | 2.19 | 9.21 | — | 5.68 | Neutral |
| T | Threonine | 2.09 | 9.10 | — | 5.87 | Neutral |
| W | Tryptophan | 2.46 | 9.41 | — | 5.88 | Neutral |
| Y | Tyrosine | 2.20 | 9.21 | 10.07 | 5.66 | Neutral/Acidic |
| V | Valine | 2.39 | 9.74 | — | 6.00 | Neutral |
When does the simple formula fail? The pI = (pKa1+pKa2)/2 formula only works for isolated free amino acids with one or two ionizable groups. For any peptide or protein with multiple residues, you must use the iterative charge-balance method because all ionizable side chains interact.
pI vs pH — What Happens Above and Below the Isoelectric Point
Understanding the relationship between pH and pI is essential for predicting protein behavior in biochemical experiments:
| pH vs pI | Net Charge | Electrophoresis | Ion Exchange |
|---|---|---|---|
| pH < pI | Positive (+) | → Cathode (−) | Binds cation exchanger |
| pH = pI | Zero (0) | Does not migrate | Minimal binding |
| pH > pI | Negative (−) | → Anode (+) | Binds anion exchanger |
In SDS-PAGE, proteins migrate only by size because SDS masks charge. In native PAGE and isoelectric focusing, the isoelectric point directly determines migration. At exactly pH = pI, a protein has zero net charge, experiences no driving force in an electric field, and is least soluble in aqueous solution.
⚠️ At the isoelectric point, protein solubility is at its minimum. This can cause aggregation and precipitation during purification — always buffer proteins away from their pI unless intentionally precipitating them.
Isoelectric Point of Common Proteins and Amino Acids — Reference Table
Common Proteins — Known Isoelectric Points
| Protein | pI | Classification | Notes |
|---|---|---|---|
| Pepsin | 1.0 | Very acidic | Gastric protease, active at low pH |
| Human Serum Albumin | 4.7 | Acidic | Major blood carrier protein |
| Ovalbumin (egg white) | 4.7 | Acidic | Precipitates when acidified |
| Casein (milk) | 4.6 | Acidic | Curdles at pH 4.6 (pI) |
| Insulin | 5.4 | Slightly acidic | Pancreatic hormone |
| Myoglobin | 7.36 | Near neutral | Oxygen storage in muscle |
| Hemoglobin | 6.8–7.0 | Near neutral | Oxygen transport in blood |
| Cytochrome c | 10.7 | Basic | Electron carrier, binds anion membranes |
| Lysozyme | 11.35 | Very basic | Antibacterial enzyme in tears/saliva |
| Histones | 10–11 | Very basic | Rich in Lys/Arg; binds negatively charged DNA |
How to Calculate Isoelectric Point for MCAT
The MCAT tests isoelectric point calculations using the simple formula method. Here are the four most commonly tested MCAT isoelectric point problem types:
MCAT Type 1 — Neutral Amino Acid pI
For any neutral amino acid (Gly, Ala, Val, Leu, Ile, Ser, Thr, Phe, Trp, Met, Asn, Gln):
pI = (pKa_carboxyl + pKa_amino) / 2
Glycine: pI = (2.35 + 9.87) / 2 = 6.11
MCAT Type 2 — Acidic Amino Acid pI
For Asp and Glu (acidic side chain): pI = (pKa_carboxyl + pKa_sidechain) / 2
Aspartate: pI = (1.99 + 3.90) / 2 = 2.95 — always below 7 for acidic amino acids
MCAT Type 3 — Basic Amino Acid pI
For Lys, Arg, His (basic side chain): pI = (pKa_amino + pKa_sidechain) / 2
Arginine: pI = (8.99 + 12.48) / 2 = 10.74 — always above 7 for Lys/Arg
Histidine: pI = (9.33 + 6.04) / 2 = 7.69 — near neutral due to low side chain pKa
MCAT Type 4 — Predicting Charge at Physiological pH
Question: "What charge does Lysine (pI = 9.60) carry at physiological pH 7.4?"
Solution: pH 7.4 < pI 9.60 → the solution is below Lysine's pI → Lysine carries a net positive charge (+1) at pH 7.4.
Rule: pH < pI → positive; pH > pI → negative; pH = pI → neutral
💡 MCAT Memory Tip: Acidic amino acids (D, E) have pI < 3 — they're negatively charged at pH 7. Basic amino acids (K, R) have pI > 9 — they're positively charged at pH 7. His (pI ≈ 7.6) is the only amino acid that can change its charge sign near physiological pH.
Worked Examples — Isoelectric Point Problems
Example 1 — Glycine (simplest case)
- Neutral amino acid; pKa1 = 2.35, pKa2 = 9.87
- pI = (2.35 + 9.87) / 2 = 6.11
- At pH 7.0: slightly negative (pH > pI 6.11)
Example 2 — Glutamate (acidic)
- Acidic: pKa1 = 2.10, pKa3 (side chain) = 4.25
- pI = (2.10 + 4.25) / 2 = 3.18
- At pH 7.4: strongly negative (pH 7.4 ≫ pI 3.18)
Example 3 — Arginine (strongly basic)
- Basic: pKa2 = 8.99, pKa3 (guanidinium) = 12.48
- pI = (8.99 + 12.48) / 2 = 10.74
- At pH 7.4: strongly positive (pH 7.4 < pI 10.74) — binds DNA and phospholipids
Example 4 — Histidine (unique: near-neutral pI)
- Basic: pKa2 = 9.33, pKa3 (imidazole) = 6.04
- pI = (9.33 + 6.04) / 2 = 7.69
- His is the only ionizable amino acid that can change protonation state near physiological pH — critical for enzyme active sites and hemoglobin buffering
Example 5 — Peptide RKRK (basic peptide)
- Ionizable groups: N-term (pKa 8.0), C-term (pKa 3.1), 2×Arg (pKa 12.48), 2×Lys (pKa 10.53)
- Sequence is rich in positive groups → pI expected to be very high
- Bisection converges to pI ≈ 11.8
- At pH 7.4: strongly positive; will bind anion exchange columns
Example 6 — Predicting ion exchange behavior
- Protein A: pI = 4.5; running at pH 7.0
- pH (7.0) > pI (4.5) → Protein A is negatively charged at pH 7.0
- Will bind DEAE (anion exchange) column; will not bind CM-cellulose (cation exchange)
- To elute from DEAE: increase salt concentration or raise pH to weaken binding