Isoelectric Point (pI) Calculator

Find the pH where peptide net charge equals zero

Contents

What is the Isoelectric Point?

The isoelectric point (pI) is the pH at which a peptide or protein carries zero net electrical charge. At this specific pH value, the sum of all positive charges exactly equals the sum of all negative charges, resulting in a neutral molecule.

⚖️

The Fundamental Definition

At pH = pI: Qpositive = Qnegative
Therefore: Qnet = 0

The term "isoelectric" comes from Greek: iso (equal) + electric (charge). At the pI, the molecule experiences no net electrostatic force in an electric field—hence it is "isoelectric."

Important: At the pI, peptides exist as zwitterions (German: zwitter = "hybrid" or "hermaphrodite"). A zwitterion is a molecule that contains both positive and negative charges simultaneously, resulting in zero net charge. The peptide still has charged groups—it's not uncharged, it's electrically neutral.

⚡ Understanding Zwitterions

A common misconception is that peptides at their pI have "no charges." This is incorrect! At the pI, peptides are zwitterionic—they carry both positive and negative charges that balance each other out:

pH < pI:
More groups protonated (+) than deprotonated (−)
Net positive charge
pH = pI (Zwitterion):
Equal positive (+) and negative (−) charges
Net charge = 0
Still has individual charges, but they cancel!
pH > pI:
More groups deprotonated (−) than protonated (+)
Net negative charge

Example: A peptide with pI = 7.0 at pH 7.0 might have:
• 3 positive charges (N-term + 2 Lys)
• 3 negative charges (C-term + 2 Asp)
• Net charge = +3 + (−3) = 0 → Zwitterion

This is why peptides at their pI still participate in electrostatic interactions—the individual charged groups interact with solvent, ions, and other molecules even though the net charge is zero.

Historical Context

The concept of the isoelectric point was introduced by Søren Sørensen in 1909, the same scientist who developed the pH scale. Sørensen discovered that proteins precipitate most readily at their pI, a phenomenon that became the foundation for numerous purification techniques still used today.

🔬 Why pI Matters
At the pI, peptides show minimum solubility, maximum aggregation tendency, and unique behavior in electric fields. Understanding pI is essential for purification, formulation, and predicting biological behavior.
📊 pI vs pH
pI is a property of the peptide (intrinsic), while pH is a property of the solution (extrinsic). Every peptide has one pI value, but can exist at any pH depending on the buffer.
⚡ Zero-Crossing Point
On a charge-pH curve, the pI is where the line crosses zero. This graphical representation makes it easy to visualize how charge changes with pH. See charge-pH curves →
🎯 Practical Impact
Knowing the pI helps select optimal pH for storage (away from pI for stability), purification (near pI for precipitation, far from pI for solubility), and chromatography (exploit charge differences).

The Significance of pI

The isoelectric point is far more than an academic curiosity—it's a critical parameter that determines peptide behavior in biological systems and laboratory applications.

Application 1: Isoelectric Focusing (IEF)

🔬 The Gold Standard for Protein Separation

Principle: In an electric field with a pH gradient, peptides migrate until they reach their pI, where they stop moving (zero net charge). This creates extremely sharp bands.

Resolution: IEF can separate proteins differing by as little as 0.01 pH units in their pI values—far superior to other electrophoresis techniques.

Applications:

  • 2D gel electrophoresis: IEF (1st dimension by pI) + SDS-PAGE (2nd dimension by size)
  • Quality control: Detecting charge variants, deamidation, oxidation
  • Capillary IEF: High-throughput analysis of therapeutic proteins
  • Preparative IEF: Large-scale purification based on pI differences

Application 2: Ion Exchange Chromatography

🧪 pH Selection for Binding and Elution

Cation exchange (negatively charged resin):
• Bind at pH < pI (peptide is positive)
• Elute at pH > pI (peptide becomes negative/neutral)

Anion exchange (positively charged resin):
• Bind at pH > pI (peptide is negative)
• Elute at pH < pI (peptide becomes positive/neutral)

Strategy example: Separating two peptides with pI = 6.5 and pI = 8.5
• Use cation exchange at pH 7.5
• Peptide 1 (pI 6.5) is negative → flows through
• Peptide 2 (pI 8.5) is positive → binds to resin

Application 3: Formulation Development

💊 Solubility and Stability Optimization

Minimum solubility at pI: Without net charge, there's no electrostatic repulsion to prevent aggregation. Peptides precipitate most readily near their pI.

Maximum aggregation at pI: Hydrophobic interactions dominate when electrostatic repulsion is absent, leading to protein-protein association.

Storage strategy:

  • Store peptides at pH far from pI (|pH - pI| > 1.5 units)
  • Target |charge| > 3 for stable liquid formulations
  • Add salt if needed to screen residual charges
  • Consider lyophilization if solution stability is poor

Example: Insulin (pI ≈ 5.3)
• Formulated at pH 7.4 (charge ≈ -2)
• Zinc added to promote controlled hexamer formation
• Stored away from pI for maximum shelf life

Application 4: Subcellular Localization

🧬 Predicting Protein Localization

Proteins in different cellular compartments tend to have characteristic pI ranges:

Acidic proteins (pI 4-5)
Cytoplasmic, extracellular
Neutral proteins (pI 6-8)
Membrane-associated, secreted
Basic proteins (pI 9-12)
Nuclear, DNA/RNA-binding

This correlation reflects the electrostatic environment and functional requirements of each compartment. DNA-binding proteins are basic (pI > 9) to interact with negatively charged phosphate backbones.

Calculating pI: The Challenge

Unlike molecular weight or extinction coefficient, which can be calculated directly from sequence, the pI requires solving an equation that has no closed-form solution. We must use iterative numerical methods to find it.

Why Is pI Calculation Difficult?

🧮 The Mathematical Challenge

To find the pI, we need to solve this equation:

Qnet(pH) = 0

where Qnet(pH) is the sum of all charge contributions calculated using Henderson-Hasselbalch:

Qnet(pH) = QN-term(pH) + QC-term(pH) + Σ Qside chains(pH)

Each term contains exponentials (10pH-pKa), making the overall equation impossible to solve algebraically. Instead, we use numerical methods like:

  • Bisection method: Simple, robust, always converges
  • Newton-Raphson: Faster but requires derivative calculations
  • Secant method: Compromise between speed and simplicity

Conceptual Approach: Finding the Zero-Crossing

Think of the pI as the "balance point" on a charge-pH curve. The curve starts positive at low pH (all groups protonated) and ends negative at high pH (all groups deprotonated). Somewhere in between, it crosses zero—that's the pI.

Start
pH range: 0 to 14
Charge at pH 0: Positive (all groups protonated)
Charge at pH 14: Negative (all groups deprotonated)
→
Iterate
Check midpoint: pH 7
If charge > 0: pI is in upper half (7-14)
If charge < 0: pI is in lower half (0-7)
→
Converge
Repeat: Narrow range each iteration
Stop when: |charge| < 0.01
Result: pI to 2 decimal places

Step-by-Step: Bisection Method

The bisection method is the most straightforward approach to finding the pI. Let's calculate the pI of melittin (GIGAVLKVLTTGLPALISWIKRKRQQ) step by step.

Example Peptide: Melittin
GIGAVLKVLTTGLPALISWIKRKRQQ
26 amino acids | 4 Lys + 3 Arg = highly basic → expect high pI

Iteration Steps

Iteration pHlow pHhigh pHmid Charge Action
1 0.00 14.00 7.00 +6.98 pI > 7.00 → pHlow = 7.00
2 7.00 14.00 10.50 +3.47 pI > 10.50 → pHlow = 10.50
3 10.50 14.00 12.25 -0.34 pI < 12.25 → pHhigh = 12.25
4 10.50 12.25 11.38 +1.82 pI > 11.38 → pHlow = 11.38
5 11.38 12.25 11.81 +0.66 pI > 11.81 → pHlow = 11.81
6 11.81 12.25 12.03 +0.14 pI > 12.03 → pHlow = 12.03
7 12.03 12.25 12.14 -0.10 pI < 12.14 → pHhigh = 12.14
8 12.03 12.14 12.09 +0.02 Converged!

Final Result:

pI (melittin) = 12.09

Interpretation: Melittin has a very high pI due to its high content of basic residues (4 Lys + 3 Arg) and lack of acidic residues. This makes it strongly positive at physiological pH, enabling it to bind to and disrupt negatively charged cell membranes.

💡 Understanding Convergence

Notice how the pH range narrows with each iteration:

  • Iteration 1: Range = 14.00 units
  • Iteration 2: Range = 7.00 units
  • Iteration 4: Range = 1.75 units
  • Iteration 8: Range = 0.11 units → |charge| < 0.01

The bisection method is guaranteed to converge because the charge function is continuous and monotonically decreasing with pH. Each iteration halves the search space.

Interactive pI Calculator

Use these calculators to compute pI values and explore how sequence and modifications affect the isoelectric point.

🧮 Calculator 1: pI from Sequence

Calculate the isoelectric point using the bisection method:

🔄 Calculator 2: pI Shift Predictor

See how mutations or modifications change the pI:

pI in Protein Purification

The isoelectric point is central to many protein purification strategies. Understanding how to exploit pI differences is essential for efficient separation.

Strategy 1: Isoelectric Precipitation

Principle

At the pI, proteins have minimum solubility and will precipitate out of solution. By adjusting pH to the target protein's pI, you can selectively precipitate it while contaminants remain soluble.

Procedure

  1. Determine target protein's pI (calculate or measure experimentally)
  2. Adjust solution pH to within 0.5 units of pI
  3. Allow time for precipitation (30 min - 2 hours)
  4. Centrifuge to collect precipitate
  5. Resolubilize at pH far from pI (|pH - pI| > 2)

When to Use

Best for bulk purification when target pI is significantly different from contaminants (ΔpI > 2 units). Commonly used for immunoglobulin purification.

Strategy 2: pH-Optimized Ion Exchange

Selecting the Right pH

The key to successful ion exchange is choosing a pH where your target protein is charged opposite to major contaminants.

Step 1: Calculate pI of target and major contaminants
Step 2: Find pH where target has strong charge (|pH - pI| > 1.5)
Step 3: Verify contaminants have opposite or weaker charge
Step 4: Select resin type based on target charge at chosen pH

Example Purification

Target: Lysozyme (pI = 11.0)
Contaminant: BSA (pI = 4.7)
Strategy: Use cation exchange at pH 8.0
• Lysozyme (pH 8.0 < pI 11.0) → Positive → Binds to resin
• BSA (pH 8.0 > pI 4.7) → Negative → Flows through

Strategy 3: Chromatofocusing

Advanced pI-Based Separation

Chromatofocusing creates a pH gradient in a column, causing proteins to focus at their pI and elute sequentially. It combines the resolution of IEF with the scalability of column chromatography.

Resolution: Can separate proteins with ΔpI as small as 0.02 pH units
Capacity: Works with mg to gram quantities
Application: Particularly useful for closely related protein variants

pI and Peptide Properties

The isoelectric point correlates with several important physical and biological properties of peptides and proteins.

💧 Solubility
Minimum at pI due to lack of electrostatic repulsion between molecules. Although peptides at pI are zwitterions (containing both + and − charges), the net charge is zero, so molecules don't repel each other. Maximum solubility occurs far from pI where net charge provides strong electrostatic repulsion. For formulation, target |pH - pI| > 1.5 units.
🔗 Aggregation
Maximum at pI because molecules in the zwitterionic state lack net charge-based repulsion. While individual charged groups still exist, the absence of net charge allows hydrophobic interactions to dominate, leading to aggregation, precipitation, or fibril formation. This can be irreversible in storage.
🧬 Membrane Binding
Peptides with pH < pI are positive and can bind to negatively charged membranes. At pH = pI, membrane affinity is minimized. Cell-penetrating peptides typically have pI > 9 to ensure positive charge at pH 7.4.
⚗️ Chemical Stability
Some degradation pathways (deamidation, isomerization) are pH-dependent. While not always at pI, there's often an optimal pH for long-term stability that balances chemical and physical degradation.
🎯 Biological Activity
Many peptide-receptor interactions are charge-dependent. The pI helps predict whether a peptide will be charged at the target site's pH (e.g., endosomal pH ~5-6 vs cytoplasmic pH ~7.4).
📊 Crystallization
Proteins often crystallize best near (but not exactly at) their pI. The reduced solubility helps nucleation, but some charge is needed to order the crystal lattice. Try pH = pI ± 0.5 units.
💡 pH-Dependent Charge and Biological Function

Remember from our discussions of net charge and acid-base chemistry: both histidine (pKa 6.04) and cysteine (pKa 8.3) are sensitive to pH changes near physiological pH.

Peptides rich in His or Cys can show pH-dependent behavior as they transition through different cellular compartments:

  • Endosome (pH 5-6): Histidine becomes protonated (+), promoting endosomal escape
  • Cytoplasm (pH 7.4): Both His and Cys are mostly neutral
  • Alkaline compartments: Cysteine becomes deprotonated (-)

This pH-sensitivity is exploited in drug delivery systems and explains why the pI alone doesn't fully predict behavior—you must consider the local pH environment.

Comparing pI Values

Different classes of proteins and peptides have characteristic pI ranges that reflect their biological roles and cellular localization.

Typical pI Ranges by Protein Class

Protein Class Typical pI Range Examples Biological Context
Acidic Proteins 4.0 - 5.5 Pepsin (1.0), BSA (4.7), Ovalbumin (4.6) Extracellular, cytoplasmic enzymes
Neutral Proteins 6.0 - 8.0 Actin (5.5), Myoglobin (7.2), Hemoglobin (6.8) Muscle proteins, oxygen carriers
Basic Proteins 9.0 - 12.0 Lysozyme (11.0), Ribonuclease (9.6), Histones (10-11) DNA-binding, nuclear proteins
Antimicrobial Peptides 9.5 - 12.5 Melittin (12.1), LL-37 (10.5), Defensins (8-10) Membrane-disrupting peptides
Cell-Penetrating Peptides 10.0 - 13.0 TAT (12.3), Penetratin (12.6) Drug delivery vehicles

Notable Peptide Examples

FLAG Tag (DYKDDDDK)

pI: 3.96 (very acidic)

Composition: 5 Asp, 1 Lys, 1 Tyr

Use: Affinity purification tag

Highly negative at physiological pH, ensuring strong binding to anti-FLAG antibodies.

Oxytocin

pI: ~7.7 (nearly neutral)

Composition: Disulfide-bridged nonapeptide with C-terminal amidation

Use: Hormone (social bonding, labor induction)

Balanced charge distribution, minimal net charge at physiological pH.

Melittin (bee venom)

pI: ~12.1 (very basic)

Composition: 4 Lys, 3 Arg, no acidic residues

Use: Membrane disruption

Strongly positive at all biological pH values, binds and disrupts membranes.

Modified Peptides & pI

Post-translational modifications and synthetic modifications can dramatically shift the pI by adding or removing ionizable groups.

Terminal Modifications

⚠️ Terminal Modifications Shift pI

As discussed in our net charge page, acetylated N-termini and amidated C-termini are NOT ionizable:

Acetylation (Ac-N-term)

Removes: +1 charge at pH < 9

Effect on pI: Decreases pI (more acidic)

Magnitude: Typically -0.5 to -2.0 pH units depending on sequence

Amidation (-NH₂ C-term)

Removes: -1 charge at pH > 3

Effect on pI: Increases pI (more basic)

Magnitude: Typically +0.5 to +2.0 pH units depending on sequence

Example: Basic peptide KKKRRR
• Unmodified pI: ~11.5
• Ac-KKKRRR pI: ~10.8 (Δ = -0.7)
• KKKRRR-NH₂ pI: ~12.2 (Δ = +0.7)
• Ac-KKKRRR-NH₂ pI: ~11.5 (changes cancel out!)

Common Post-Translational Modifications

Phosphorylation
Decreases pI
Residues: Ser, Thr, Tyr
Charge: Adds -2 at physiological pH (phosphate group, pKa ~2 and ~7)
ΔpI: -0.5 to -1.5 per phosphorylation
Detection: pI shift used in 2D gels to detect phosphorylation
Methylation
Increases pI (slightly)
Residues: Lys, Arg
Charge: Preserves +1 but increases pKa slightly
ΔpI: +0.1 to +0.3 per methylation
Note: Trimethylation has larger effect than mono/dimethylation
Acetylation (Lys)
Decreases pI
Residues: Lys sidechains
Charge: Removes +1 (makes Lys neutral)
ΔpI: -0.5 to -2.0 per acetylation
Function: Histone acetylation reduces DNA binding
Deamidation
Decreases pI
Residues: Asn → Asp, Gln → Glu
Charge: Converts neutral to -1
ΔpI: -0.3 to -0.8 per deamidation
Note: Spontaneous degradation in storage
Glycosylation
Variable (usually decreases pI)
Residues: Asn (N-glyc), Ser/Thr (O-glyc)
Charge: Depends on sugar composition (sialic acid = -1)
ΔpI: 0 to -2.0 depending on glycan structure
Note: Highly variable, affects IEF patterns
Sulfation
Decreases pI
Residues: Tyr
Charge: Adds -1 (sulfate group, pKa ~2)
ΔpI: -0.5 to -1.0 per sulfation
Example: Sulfated CCK peptides

🚀 PepDraw Pro: Modification Support

Future versions of PepDraw will include built-in support for common modifications:

✓ Phosphorylation (pSer, pThr, pTyr)
✓ Methylation (meLys, meArg)
✓ Acetylation (acLys)
✓ Automatic pI recalculation with modifications

Common Mistakes & Tips

Avoid these frequent errors when calculating and interpreting pI:

❌ Confusing pI with pH
Mistake: Treating pI as if it's a pH value that can be "set" experimentally.

Reality: pI is an intrinsic property of the peptide (like molecular weight). pH is a property of the solution. You can't "change" a peptide's pI without modifying its sequence.

Correct thinking: "At what pH does my peptide reach its pI?" not "What's the pI of my buffer?"
❌ Thinking "pI = No Charges"
Mistake: Believing peptides at pI have no charged groups.

Reality: At pI, peptides are zwitterions—they have both positive and negative charges that balance to zero net charge. The individual charges are still present and active!

Example: A peptide at pI might have +5 charges and -5 charges simultaneously (net = 0), not zero total charges. This is why they still interact electrostatically with solvent and ions even at pI.
❌ Storing Proteins at pI
Mistake: Formulating peptides at their pI for "stability."

Problem: At pI, solubility is minimum and aggregation is maximum. This causes precipitation!

Fix: Store at pH far from pI (|pH - pI| > 1.5 units) where charge provides stabilizing electrostatic repulsion.
❌ Ignoring Terminal Modifications
Mistake: Using unmodified pI calculation for Ac- or -NH₂ modified peptides.

Impact: Can shift pI by 0.5-2.0 units, completely changing purification strategy.

Fix: Always check for terminal modifications before calculating pI. PepDraw's calculator accounts for these automatically.
❌ Assuming pI = Optimal Crystallization pH
Mistake: Trying to crystallize exactly at pI.

Reality: Proteins often crystallize best near pI (±0.5 units) but not exactly at it.

Strategy: Screen pH around pI ± 0.5 to find the sweet spot between reduced solubility and ordered packing.
✅ Use pI to Predict Charge Sign
Tip: Quick rule to know if your peptide is positive or negative:

If pH < pI: Peptide is positive
If pH > pI: Peptide is negative
If pH = pI: Peptide is neutral (zero net charge)

This helps select the right chromatography resin without detailed calculations!
✅ Verify pI Experimentally with IEF
Tip: Calculated pI is an estimate. For critical applications, measure experimentally using isoelectric focusing.

Why: pKa values can shift in folded proteins, modifications may be present, or sequence errors exist.

When to verify: Therapeutic proteins, difficult purifications, unexplained precipitation issues.
✅ Consider Local pH Microenvironments
Tip: The pH near membranes, in active sites, or in organelles can differ from bulk pH by 1-2 units.

Example: Near negatively charged membranes, local pH is ~0.5 units lower than bulk due to proton accumulation.

Impact: A peptide with pI 7.5 might be neutral in bulk pH 7.4 but positive near membranes (local pH ~6.9).
✅ Use pI Shift to Detect Degradation
Tip: Changes in pI over time indicate chemical degradation.

Common causes:
• Deamidation (Asn→Asp, Gln→Glu) → pI decreases
• Oxidation of Met/Trp → variable
• Cleavage → multiple pI values appear

Monitoring: IEF is more sensitive than mass spec for detecting charge variants.

References

1. Historical Foundation
Sørensen, S. P. L. (1909). Enzyme studies II: The measurement and significance of hydrogen ion concentration in enzymatic processes. Biochemische Zeitschrift, 21, 131-304.
2. Textbook Reference
Nelson, D. L., & Cox, M. M. (2017). Lehninger Principles of Biochemistry (7th ed.). W. H. Freeman. Chapter 3: Amino Acids, Peptides, and Proteins.
3. pKa Values and pI Calculation
Bjellqvist, B., Hughes, G. J., Pasquali, C., Paquet, N., Ravier, F., Sanchez, J. C., ... & Hochstrasser, D. F. (1993). The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis, 14(1), 1023-1031. doi:10.1002/elps.11501401163
4. Computational Tools
Gasteiger, E., Hoogland, C., Gattiker, A., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook (pp. 571-607). Humana press. doi:10.1385/1-59259-890-0:571
5. Isoelectric Focusing Methodology
Righetti, P. G., Stoyanov, A. V., & Zhukov, M. Y. (2001). The Proteome Revisited: Theory and Practice of All Relevant Electrophoretic Steps. Elsevier. ISBN: 978-0444505750
6. Protein Purification Strategies
Scopes, R. K. (1994). Protein Purification: Principles and Practice (3rd ed.). Springer-Verlag. Chapter 5: Separation by Charge. ISBN: 978-0387940724

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