18,575
M⁻¹cm⁻¹
6.192
0.619
11,000
M⁻¹cm⁻¹
7,450
M⁻¹cm⁻¹
125
M⁻¹cm⁻¹
18,575
M⁻¹cm⁻¹
6.192
0.619
11,000
M⁻¹cm⁻¹
7,450
M⁻¹cm⁻¹
125
M⁻¹cm⁻¹
The Molar Extinction Coefficient Calculator estimates the UV absorbance properties of a protein at 280 nm based on its aromatic amino acid content. The extinction coefficient (ε₂₈₀) is an essential parameter for determining protein concentration using UV spectrophotometry, the most common and rapid method for quantifying purified proteins in the laboratory.
This calculation uses the Pace method, which sums the contributions of tryptophan (Trp), tyrosine (Tyr), and cystine (disulfide-bonded cysteine pairs) residues. Each of these chromophores absorbs UV light at 280 nm with characteristic molar absorptivities. The resulting extinction coefficient allows conversion of absorbance readings to molar or mass concentrations via the Beer-Lambert law.
The molar extinction coefficient at 280 nm is calculated using the Pace method (Gill and von Hippel, adjusted by Pace et al.):
$$\varepsilon_{280} = (n_{Trp} \times 5500) + (n_{Tyr} \times 1490) + (n_{Cystine} \times 125)$$
where $$n_{Trp}$$ is the number of tryptophan residues (each contributing 5500 M⁻¹cm⁻¹), $$n_{Tyr}$$ is the number of tyrosine residues (each contributing 1490 M⁻¹cm⁻¹), and $$n_{Cystine}$$ is the number of disulfide bonds (each contributing 125 M⁻¹cm⁻¹). Note that cystine refers to disulfide-bonded cysteine pairs, not free cysteine residues.
The mass extinction coefficient (absorptivity) is derived as:
$$A_{280}^{1\text{mg/mL}} = \frac{\varepsilon_{280}}{MW}$$
where MW is the molecular weight in Daltons. This gives the expected absorbance of a 1 mg/mL protein solution in a 1-cm cuvette.
A protein with ε₂₈₀ = 0 contains no Trp, Tyr, or Cystine and cannot be quantified by UV280 absorbance. In such cases, use the Bradford assay, BCA assay, or absorbance at 205 nm instead. Typical ε₂₈₀ values range from 5,000 to 200,000 M⁻¹cm⁻¹ depending on protein size and aromatic content.
The Pace method assumes the protein is fully denatured (6 M guanidinium HCl). For native proteins, the actual extinction coefficient may differ by up to 10% due to the local environment of aromatic residues affecting their absorption properties. For the most accurate concentration determination, measure absorbance under denaturing conditions or apply a correction factor.
Inputs
Results
BSA has 2 Trp, 20 Tyr, and 17 disulfide bonds. The calculated ε₂₈₀ of 42,925 M⁻¹cm⁻¹ is close to the experimentally measured value of ~43,824 M⁻¹cm⁻¹.
Inputs
Results
Lysozyme is Trp-rich (6 residues), giving a high ε₂₈₀ of 37,970 M⁻¹cm⁻¹. Its A₂₈₀ for 1 mg/mL is ~2.66, consistent with its well-characterized spectroscopic properties.
These are the only amino acids that absorb significantly at 280 nm. Tryptophan has the strongest absorption due to its indole ring system, tyrosine absorbs through its phenol group, and cystine (disulfide bonds) contributes weakly. Phenylalanine absorbs at 257 nm, not significantly at 280 nm, and is therefore excluded.
Cysteine is the free (reduced) amino acid with a thiol (-SH) group that does not absorb at 280 nm. Cystine is the oxidized form consisting of two cysteines linked by a disulfide bond (-S-S-), which absorbs weakly at 280 nm (125 M⁻¹cm⁻¹ per bond). Only count disulfide bonds, not total cysteine residues.
For denatured proteins, the Pace method is accurate to within 2-5%. For native proteins, accuracy drops to 5-15% because the local environment of aromatic residues (hydrogen bonding, solvent exposure, nearby charges) affects their absorption properties. The method is most reliable for proteins with multiple Trp residues.
If a protein lacks both Trp and Tyr, its ε₂₈₀ will be very low (only from cystine contributions) or zero. UV280 spectrophotometry is unreliable for such proteins. Alternative methods include BCA assay, Bradford assay, absorbance at 205 nm (peptide bond absorption), or amino acid analysis.
Yes, the Pace method applies to any polypeptide. However, short peptides (fewer than 20 residues) may have only 0-1 aromatic residues, giving very low ε₂₈₀ values with high relative error. For peptides, consider measuring absorbance at 214 nm or using a mass-based assay.
The Beer-Lambert law relates absorbance to concentration: A = ε × c × l, where A is absorbance (unitless), ε is the molar extinction coefficient (M⁻¹cm⁻¹), c is concentration (M), and l is path length (cm). Rearranging gives concentration: c = A/(ε × l).
In the native protein, aromatic residues may be buried in the hydrophobic core or involved in hydrogen bonding, altering their electronic environment and absorption properties. Denaturation exposes all residues to solvent, giving consistent absorption. The difference (typically 5-10%) is called the solvent perturbation effect.
Nucleic acids absorb strongly at 260 nm and significantly at 280 nm. The A260/A280 ratio for pure protein is ~0.57. If A260/A280 exceeds 0.7, nucleic acid contamination is present. Use the Warburg-Christian correction: Protein (mg/mL) = 1.55 × A₂₈₀ - 0.76 × A₂₆₀.
Standard cuvettes have a 1-cm path length. Microvolume spectrophotometers like NanoDrop use shorter path lengths (0.05-1 mm) and automatically correct to 1-cm equivalent readings. Always verify the path length setting when comparing measurements across instruments.
Yes. Modifications near aromatic residues can alter absorption. Tyrosine phosphorylation slightly shifts the absorption spectrum. Tryptophan oxidation reduces its absorption. Attached chromophores (heme, flavin, pyridoxal) add additional absorption features. For modified proteins, experimental determination of ε₂₈₀ is recommended.
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