Protein Isoelectric Point pI and Molecular Weight Tool
Compute theoretical polypeptide molecular weight and isoelectric point (pI) by summing anhydrous residue masses and solving the net-charge root across ionizable side chains and termini. The workspace opens the 238-residue green fluorescent protein reference sequence; a translated open reading frame can be pasted in its place, with unfolded pKa assumptions and post-translational modifications kept explicit.
Uses the versioned GFP example sequence (UniProtKB P42212) to illustrate residue mass summation with ProtParam average residue masses and the theoretical pI with the Bjellqvist pKa set as ExPASy ProtParam applies it.
Connect reading-frame translation to exact residue mass accounting
Before computing physicochemical properties of a protein, the exact boundary of the coding sequence must be established. Selecting a different start codon, reading frame, or stop-codon readthrough policy changes both the chain length and the terminal residue identity. Once the polypeptide string is fixed, theoretical molecular weight is calculated by summing the average isotopic masses of the anhydrous amino-acid residues and adding 18.015 Da for the free N-terminal hydrogen and C-terminal hydroxyl group.
When comparing predicted mass against sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or mass spectrometry, distinguish average isotopic mass from monoisotopic mass and account for processing events. Cleavage of an N-terminal methionine (−131.19 Da) or export signal peptide, disulfide bond formation, and affinity tag attachment alter the physical species in the sample tube relative to the raw conceptual translation of a genomic window.
Solve Henderson-Hasselbalch charge balance under explicit pKa assumptions
The theoretical isoelectric point (pI) is the unique pH at which the net electrical charge Z(pH) of the polypeptide equals zero. Because Z(pH) decreases monotonically with increasing pH—Summing fractional protonation of the N-terminus, lysine, arginine, and histidine against fractional deprotonation of the C-terminus, aspartate, glutamate, cysteine, and tyrosine—the root can be bracketed to arbitrary numerical precision by bisection.
Numerical precision in the root solver should not be confused with experimental accuracy. Because published pKa tables (such as Bjellqvist immobilized-gradient constants versus classical text constants) assign slightly different values to terminal amines, carboxylates, and histidine imidazoles, two tools can differ by 0.1 to 0.3 pH units on the same sequence. Use theoretical pI as an initial guide for ion-exchange buffer selection or solubility screening, and record the chosen pKa convention alongside the construct sequence.
Equations and variables
Anhydrous polypeptide molecular weight
MW = Σi ni Mi,res + M(H2O)
- MW
- Theoretical unmodified monomeric polypeptide molecular mass (Da)
- ni, Mi,res
- Count and anhydrous peptide-bond residue mass of amino acid i (Da)
- M(H2O)
- Terminal H and OH mass addition (18.015 Da) for a single linear chain (Da)
Polypeptide net charge as a function of pH
Z(pH) = Σpos npos / (1 + 10^(pH − pKa,pos)) − Σneg nneg / (1 + 10^(pKa,neg − pH))
- Z(pH)
- Net elementary charge of the denatured polypeptide at the stated pH
- npos, pKa,pos
- Counts and dissociation constants of N-terminus, Lys, Arg, and His groups
- nneg, pKa,neg
- Counts and dissociation constants of C-terminus, Asp, Glu, Cys, and Tyr groups
Scope and limitations
- Theoretical pI calculations use intrinsic pKa values measured on unfolded model peptides and ignore 3D electrostatic coupling, buried side chains, and bound cofactors.
- Polypeptide mass sums assume a single unmodified linear chain without disulfide bond proton loss (−2.016 Da per cystine bridge), glycosylation, phosphorylation, or signal-peptide cleavage.
- Different published pKa scales (such as Bjellqvist, Rodwell, or Solomon) yield slightly different theoretical pI values for the same amino-acid sequence.
Sources and provenance
- UniProtKB P42212 green fluorescent proteinCC BY 4.0
- Bjellqvist et al. 1993 polypeptide isoelectric points in immobilized pH gradientsArticle citation; reference pKa scale methodology
- IUPAC standard atomic weights of the elements 2021Cited atomic-weight constants; used by the alternative CIAAW residue table
Technical questions
Why do theoretical pI values often differ from native isoelectric focusing measurements?
Sequence-based pI solvers treat every ionizable residue as fully solvent-exposed with an unperturbed model-peptide pKa. In a folded native protein, salt bridges, hydrophobic burial, and local dielectric microenvironments shift individual side-chain pKa values by up to several pH units.
Why is 18.015 Da added to the sum of amino-acid residue masses?
Each internal peptide bond forms by condensation, losing one water molecule (18.015 Da) relative to free amino acids. Summing anhydrous residue masses across a chain of length N leaves one uncondensed H at the N-terminus and one OH at the C-terminus, requiring a single +18.015 Da terminal correction per polypeptide chain.
How do disulfide bonds or N-terminal formylation change the calculated mass and pI?
Each oxidized disulfide bond removes two hydrogen atoms (−2.016 Da) and eliminates two titratable thiol groups (Cys pKa 9.0 in the Bjellqvist set the engine uses) from the net-charge equation. Similarly, N-terminal acetylation or formylation blocks the positively charged alpha-amino group, shifting the isoelectric point toward more acidic values.
Connected workflows
- Six-frame translation and ORF inspection
Identify the exact start and stop coordinates of candidate reading frames.
- Protein A280 concentration calculation
Use molecular weight and extinction coefficients for A280 concentration checks.
- EGFR sequence and structure inspection
Compare primary sequence coordinates with 3D structural domain coverage.