Extinction coefficient calculator

What is an extinction coefficient?

The molar extinction coefficient ($\varepsilon$) describes how strongly a protein absorbs light at a given wavelength. At 280 nm, sequence-based protein estimates come mainly from tryptophan, tyrosine, and cystine, the disulfide-bonded form of cysteine.

This calculator uses the protein sequence to estimate $\varepsilon_{280}$, molecular weight, predicted $A_{280}$ for a 0.1% solution, and optional concentration from a measured absorbance value. It is intended for purified proteins without additional UV-absorbing cofactors, labels, nucleic acids, or other chromophores.

For a broader physicochemical profile, use Protein Parameters, which reports molecular weight, pI, amino acid composition, instability index, GRAVY, and related sequence properties.

How the calculation works

The calculation follows the Pace et al. 1995 280 nm coefficients used by common protein-parameter tools:

Free reduced cysteine contributes 0 at 280 nm. A disulfide-bonded cysteine pair contributes 125, so cystine is counted per disulfide bond rather than per cysteine residue.

Molecular weight is calculated from average residue masses, subtracting peptide-bond water and adding terminal water. The Abs 0.1% value is calculated as:

where $MW$ is molecular weight in daltons. A 0.1% protein solution is 1 mg/mL, or 1 g/L.

Input format

Paste one or more canonical protein sequences as FASTA:

>Protein1
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
>Protein2
GIVEQCCTSICSLYQLENYCN

Plain one-letter protein sequence input is also accepted for a single sequence. Uploaded files can use .fasta, .fa, .fas, .txt, or .csv.

CSV input is supported when the header includes a recognizable sequence column such as sequence, seq, or protein:

id,sequence
p1,MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
p2,GIVEQCCTSICSLYQLENYCN

Only the 20 canonical amino acid letters are accepted:

ARNDCQEGHILKMFPSTWYV

Whitespace and line numbers are ignored. Punctuation, alignment gaps, stop codons, and noncanonical residue codes such as B, J, O, U, X, and Z are rejected instead of being silently removed.

Settings

If Specified disulfide coefficient is selected as the concentration basis without using Specified disulfide count as the cysteine assumption, the calculator falls back to the oxidized coefficient.

Results

The standard result table includes one row per sequence:

Optional columns are only shown when they have data:

The concentration calculation is:

where $c$ is molar concentration, $A_{280}$ is measured absorbance, $\varepsilon$ is the selected extinction coefficient, $l$ is path length in cm, and $d$ is dilution factor.

Warnings and interpretation

Warnings are shown above the result table when a result needs extra context:

The reduced and oxidized values are theoretical assumptions, not structural predictions. The calculator does not infer real disulfide bonding from a structure or sequence motif.

Accuracy considerations

Sequence-based extinction coefficients work best for purified proteins whose 280 nm absorbance is dominated by Trp and Tyr. Results can differ from experimental values because of folding state, pH, buffer composition, detergents, reducing agents, cofactors, prosthetic groups, nucleic acid contamination, labels, post-translational modifications, aggregation, and light scattering.

For quantitative assays, blank the instrument with the matching buffer, measure within the detector's linear range, and use the same cysteine assumption that matches the sample state. For high-accuracy work, determine the extinction coefficient empirically under the assay conditions.

Reference

• Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. "How to measure and predict the molar absorption coefficient of a protein." Protein Science 4, 2411-2423 (1995).