Protein molecular weight and pI calculator

What the calculator gives you

Paste a protein sequence in one-letter code and choose Calculate. FASTA headers, spaces, position numbers and one final stop (*) are ignored. The result shows the molecular weight, the monoisotopic mass, the isoelectric point (pI), the net charge at the pH you choose, the extinction coefficient at 280 nm, the absorbance of a 1 mg/mL solution, the GRAVY hydropathy score and the amino acid composition. The chart plots the net charge from pH 0 to 14.

Every value is computed the way Biopython 1.85's ProtParam module computes it, following the methods the ExPASy ProtParam documentation describes. The calculations run in your browser; the sequence is not uploaded. To get a protein sequence from DNA first, use DNA to protein.

How the molecular weight is calculated

A protein's average molecular weight is the sum of its amino acids' average masses, minus one water (18.0153 Da) for each peptide bond that joins them. A chain of n amino acids has n − 1 peptide bonds. The monoisotopic mass uses each element's most abundant isotope instead, the value mass spectrometry reports for small peptides.

The mass counts every cysteine as free. Each disulfide bond removes two hydrogen atoms, lowering the mass by 2.016 Da. Signal peptides, modifications such as phosphorylation or glycosylation, and bound metals or cofactors are not included: paste the mature chain and add those yourself.

Isoelectric point and net charge

The isoelectric point is the pH at which the protein carries no net charge. The calculator adds up the fraction of each ionizable group that is charged at a given pH, using the Henderson–Hasselbalch equation. The groups are the N-terminal amino group, the C-terminal carboxyl group and the side chains of lysine, arginine, histidine, aspartic acid, glutamic acid, cysteine and tyrosine. It then finds the pH where the charges cancel.

The pK values are those of Bjellqvist and colleagues (1993, 1994), with separate values for some terminal residues, derived from where polypeptides focus in immobilized pH gradients. A computed pI is an estimate for an unfolded chain. In a folded protein, neighboring groups shift pK values, so a measured pI can differ by more than a pH unit. The amino acid chart lists the pK values of the free amino acids, which differ from these.

Extinction coefficient and A280

Proteins absorb light at 280 nm mainly through tryptophan and tyrosine, and a little through cystines (pairs of cysteines joined by a disulfide bond). The calculator uses the coefficients of Pace and colleagues (1995):

ε₂₈₀ = 5,500 × Trp + 1,490 × Tyr + 125 × cystines, in M⁻¹ cm⁻¹.

When the sequence has two or more cysteines, the result shows ε with every pair of cysteines as a cystine, and the value if all are reduced. Choose the one that matches your protein. Dividing ε by the molecular weight gives the absorbance of a 1 mg/mL solution in a 1 cm cuvette (often written A280 0.1%). To turn a measured A280 into a concentration, use the protein concentration calculator on Plasmid Map.

Hydropathy (GRAVY)

GRAVY, the grand average of hydropathy, is the mean Kyte–Doolittle hydropathy of all the amino acids in the sequence. Above zero, the sequence is hydrophobic on average; below zero, hydrophilic. Kyte and Doolittle made the scale to find stretches of high hydropathy, which can mark a membrane-spanning segment. The amino acid chart lists each amino acid's value.

Worked example: hen egg-white lysozyme

The example is the mature chain of hen egg-white lysozyme, residues 19–147 of UniProt P00698. Residues 1–18, the signal peptide, are removed in the mature enzyme.

Lysozyme has 18 basic amino acids (6 lysines, 11 arginines, 1 histidine) against 9 acidic ones (7 aspartic acids, 2 glutamic acids), so it is positively charged at neutral pH. Its measured pI is about 11 (OpenStax Organic Chemistry, section 26.2), higher than the 9.32 computed from its sequence: an example of how folding shifts pK values.

What the calculator accepts

The 20 standard amino acids only. Ambiguous letters (B, Z, X) have no single mass, and selenocysteine (U) and pyrrolysine (O) are outside the calculator's tables, so a sequence containing any of them is refused with the letters named. Sequences can be up to 50,000 amino acids.

Sources: Biopython 1.85 ProtParam and IsoelectricPoint; the ExPASy ProtParam documentation. Automated tests compare every value with Biopython 1.85 on 157 sequences; see sources and methods.