Protein Extinction Coefficient Calculator
Estimates a protein's molar extinction coefficient at 280 nm from its tryptophan, tyrosine, and cysteine residue counts. Used to calculate protein concentration from UV absorbance measurements.
About this calculator
Proteins absorb ultraviolet light at 280 nm primarily due to three aromatic or sulfur-containing residues: tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys) when in disulfide bonds. The molar extinction coefficient ε (in M⁻¹cm⁻¹) is estimated using the Pace formula: ε = (Trp × 5500) + (Tyr × 1490) + (Cys × 125). Each coefficient reflects how strongly each residue type absorbs at 280 nm. Tryptophan is the dominant contributor with an individual extinction of 5500 M⁻¹cm⁻¹, followed by tyrosine at 1490 M⁻¹cm⁻¹, and cysteine (as disulfide) at 125 M⁻¹cm⁻¹. Once ε is known, Beer-Lambert law (A = ε × c × l) allows you to convert a spectrophotometer absorbance reading directly into molar protein concentration, making this an essential tool in biochemistry labs.
How to use
Consider a protein with 3 tryptophan residues, 5 tyrosine residues, and 2 cysteine residues (as disulfide bonds). Apply the formula: ε = (3 × 5500) + (5 × 1490) + (2 × 125) = 16500 + 7450 + 250 = 24200 M⁻¹cm⁻¹. This extinction coefficient can then be used with Beer-Lambert law. If the protein solution gives an absorbance (A) of 0.484 at 280 nm in a 1 cm cuvette, then concentration c = A / (ε × l) = 0.484 / 24200 = 2.0 × 10⁻⁵ M, or 20 μM.
Frequently asked questions
How accurate is the Pace formula for estimating protein extinction coefficients?
The Pace formula is generally accurate to within 5% for most globular proteins when the disulfide bond status of cysteines is correctly accounted for. It assumes all cysteines are in disulfide bonds; if the protein is fully reduced, you should use 0 for cysteine. For proteins lacking tryptophan, accuracy decreases because tyrosine absorbance is more sensitive to the local environment. Experimental validation using amino acid analysis is recommended for high-precision work.
Why is protein extinction coefficient measured at 280 nm instead of other wavelengths?
280 nm is chosen because tryptophan, tyrosine, and disulfide-bonded cysteine have strong, specific absorbance peaks near this wavelength, while most buffers, salts, and common laboratory reagents do not. This makes 280 nm practical and non-destructive for routine concentration measurements. By contrast, 260 nm is dominated by nucleic acid absorbance, and shorter wavelengths see too many interfering compounds.
What is the difference between molar extinction coefficient and mass extinction coefficient for proteins?
The molar extinction coefficient (ε) has units of M⁻¹cm⁻¹ and relates absorbance to molar concentration. The mass extinction coefficient (sometimes called specific absorbance, A₂₈₀¹%) relates absorbance to concentration in g/L or mg/mL, and is calculated by dividing ε by the protein's molecular weight in g/mol. The molar form is more precise for known proteins, while the mass form is convenient for rough estimates when only molecular weight is known.