Calculator
Example data table
| Example | Sequence or counts | Settings | Expected use |
|---|---|---|---|
| Short peptide | ACDEHKR | Free termini, no phosphate | Teaching pH charge balance |
| Serum protein fragment | MKWVTFISLLFLFSSAYSRGVFRR | Common protein pKa set | Fast laboratory estimate |
| Phosphoprotein region | DDEESSSTKRR | Three phosphate groups | Modified protein comparison |
| Manual count model | D 12, E 9, K 7, R 4, H 2 | Blocked termini | Large protein approximation |
Formula used
The calculator estimates net protein charge with Henderson-Hasselbalch style terms. Positive groups use this expression:
Positive charge = count / (1 + 10^(pH - pKa))
Negative groups use this expression:
Negative charge = count / (1 + 10^(pKa - pH))
Net charge is found by subtracting total negative charge from total positive charge. The isoelectric point is the pH where net charge is zero, or closest to zero.
Basic groups include the N terminus, histidine, lysine, and arginine. Acidic groups include the C terminus, aspartate, glutamate, cysteine, tyrosine, and phosphate groups.
How to use this calculator
- Paste a protein sequence in one-letter amino acid format.
- Choose sequence mode for automatic residue counting.
- Choose manual mode when only ionizable group counts are known.
- Select a pKa preset or edit each pKa value.
- Set terminal blocking, disulfide pairs, and phosphorylation count.
- Enter a target pH to view net charge at that pH.
- Press Calculate to show the result above the form.
- Use CSV or PDF buttons to save the calculation.
Protein pI calculation guide
What protein pI means
The isoelectric point is the pH where a protein has no overall charge. It does not mean every group is neutral. Some groups still carry charge. Their positive and negative values simply balance. This value helps predict solubility, migration, binding, and separation behavior.
Why sequence matters
A protein sequence contains acidic and basic residues. Aspartate and glutamate usually lower the pI. Lysine and arginine usually raise it. Histidine changes strongly near neutral pH. Cysteine and tyrosine matter more at higher pH. Terminal groups also affect short peptides.
Advanced options
This tool supports sequence parsing and manual residue counts. It also supports terminal blocking. Acetylated N termini lose positive charge. Amidated C termini lose negative charge. Disulfide bonds reduce free cysteine groups. Phosphate groups add extra acidic behavior. These options make the estimate more flexible.
How the estimate works
The calculator applies pKa values to every ionizable group. It calculates charge repeatedly from pH zero to fourteen. Then it searches for the point where charge crosses zero. A bisection method refines the answer. If no exact crossing appears, the closest charge is reported.
Using the result
The result is best treated as an estimate. Real protein pI can shift with folded structure. Salt concentration can also change behavior. Neighboring residues may alter local pKa values. Experimental methods may give different values. Still, calculated pI is useful for planning buffers. It also helps compare variants and fragments. Use the charge table to see pH trends. Export the result for reports, records, or lab notes.
FAQs
What is protein pI?
Protein pI is the pH where the calculated net charge is zero. At that pH, positive and negative ionizable groups balance each other.
Can I paste a FASTA sequence?
Yes. The calculator ignores non-letter characters. It keeps valid uppercase amino acid letters and counts the residues needed for charge estimation.
Which residues affect pI most?
Aspartate, glutamate, histidine, lysine, and arginine often have the largest effect. Cysteine, tyrosine, termini, and modifications can also matter.
Why do pKa presets change the answer?
Different references use different pKa assumptions. Small pKa changes can shift the net charge curve and move the estimated pI.
Should I include terminal groups?
Include them for normal free proteins and peptides. Block them when the N terminus or C terminus is chemically modified.
How are disulfide bonds handled?
Each disulfide pair removes two free cysteine side chains from the ionizable cysteine count. This prevents bonded cysteines from acting as free thiols.
How are phosphate groups handled?
Each phosphate group is treated as a diprotic acidic group. It can add up to two negative charge units depending on pH.
Is this result experimental pI?
No. It is a calculated estimate. Folding, salts, nearby residues, and experimental conditions can shift the measured protein pI.