Extinction coefficient calculator

What is the extinction coefficient?

The molar extinction coefficient ($\varepsilon$) quantifies how strongly a protein absorbs light at a specific wavelength. At 280 nm, proteins absorb UV light primarily through their aromatic amino acids—tryptophan, tyrosine, and cystine (disulfide-bonded cysteines).

This value is essential for determining protein concentration using UV-Vis spectroscopy. By measuring absorbance at 280 nm and applying the Beer-Lambert law, you can calculate precise protein concentrations without destructive assays.

For a comprehensive analysis that includes extinction coefficient along with molecular weight, isoelectric point, and other physicochemical properties, use our Protein Parameters calculator.

How to calcualte extinction coefficient?

This tool uses the Pace method (Pace et al., 1995) to calculate the theoretical molar extinction coefficient from the amino acid sequence. The calculation is based on the additive contributions of the three UV-absorbing residues.

The Pace equation

The molar extinction coefficient at 280 nm is calculated using:

$$\varepsilon_{280} = (n_{Trp} \times 5500) + (n_{Tyr} \times 1490) + (n_{Cys} \times 125)$$

Where:

• $n_{Trp}$: Number of tryptophan residues (W)
• $n_{Tyr}$: Number of tyrosine residues (Y)
• $n_{Cys}$: Number of cysteine residues (C)

Contribution coefficients

Each amino acid contributes a fixed amount to the overall extinction coefficient:

Tryptophan dominates UV absorption due to its indole ring system. Tyrosine contributes through its phenol group. Cysteine contributes only when forming disulfide bonds (cystine).

Accuracy considerations

The Pace method is accurate to approximately $\pm 5\%$ for most globular proteins. The actual extinction coefficient can vary depending on the protein's three-dimensional structure and the buffer environment.

Proteins lacking tryptophan residues will have significantly lower extinction coefficients and may be difficult to quantify reliably at 280 nm.

Input requirements

• Protein sequences: Enter one or more protein sequences in FASTA format
• Supports batch processing of multiple sequences
• Accepts .fasta, .fa, .fas, and .txt file uploads
• Can fetch sequences directly from RCSB PDB

Understanding the results

The output table includes:

• Protein ID: Identifier from the FASTA header
• Number of Amino Acids: Total sequence length
• Extinction Coefficient: Molar extinction coefficient in $\text{M}^{-1}\text{cm}^{-1}$

Using the extinction coefficient

To calculate protein concentration from an absorbance measurement, use the Beer-Lambert law:

$$c = A_{280} / (\varepsilon \cdot l)$$

Where:

• $c$: Concentration (M)
• $A_{280}$: Measured absorbance at 280 nm
• $\varepsilon$: Extinction coefficient ($\text{M}^{-1}\text{cm}^{-1}$)
• $l$: Path length (cm), typically 1 cm

For concentration in mg/mL, multiply the molar concentration by the molecular weight (in Da) and divide by 1000.

Use cases

The extinction coefficient is commonly needed for:

• Determining protein concentration from UV absorbance measurements
• Planning protein purification chromatography experiments
• Calculating dilution factors for spectroscopic assays
• Validating protein expression and yield
• Quality control in protein production workflows

Limitations

The Pace method works well for most globular proteins, but has known limitations:

• Assumes all cysteine residues are in reduced form (not disulfide-bonded)
• Does not account for non-standard amino acids or post-translational modifications
• Buffer conditions and protein folding state can affect actual absorbance
• Proteins without tryptophan may have extinction coefficients too low for accurate $A_{280}$ measurements

Related tools

• Protein Parameters — Calculates extinction coefficient along with molecular weight, pI, GRAVY, instability index, and other properties in a single analysis
• Molecular Weight — Calculate protein molecular weight for converting molar concentration to mg/mL
• isoelectric point — Determine protein pI for buffer selection and purification planning
• Amino Acid Composition — View the full amino acid breakdown including Trp, Tyr, and Cys counts

Based on: Pace, C.N., et al. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Science, 4(11), 2411-2423. DOI: 10.1002/pro.5560041120