Protein Extinction Coefficient at 280 nm

Relate extinction coefficient to absorbance

At 280 nm, the molar extinction coefficient ε describes how strongly a molecule absorbs light per unit concentration and optical path length. A spectrophotometer measures absorbance A₂₈₀, a unitless reading that depends on both the sample concentration and the optical path length.

The Beer–Lambert law gives A₂₈₀ = ε × c × ℓ. Use ε in M⁻¹·cm⁻¹, concentration c in mol/L, and optical path length ℓ in cm. To estimate concentration from a blank-corrected reading, rearrange the equation to c = A₂₈₀ / (ε × ℓ), using an extinction coefficient appropriate for the sample.

Work through the calculator example

Load WYCCC in the calculator. It contains 1 tryptophan (W), 1 tyrosine (Y), and 3 cysteines (C). In the reduced case, the model counts contributions from W and Y only, giving 5,500 + 1,490 = 6,990 M⁻¹·cm⁻¹.

If two cysteines form one disulfide bond within this chain, the pair is called cystine. The third cysteine stays unpaired. The model adds 125 M⁻¹·cm⁻¹ for the pair, giving 7,115 M⁻¹·cm⁻¹.

For a sample at 100 µM (0.0001 mol/L) with a 1 cm path length, multiply each coefficient by 0.0001 × 1 to get the expected absorbance.

Compare the estimate with a measurement

The two values describe assumed cysteine states within one chain. Establishing which disulfides actually formed requires independent evidence. When comparing with a measured A₂₈₀, use the instrument’s effective path length and a matched buffer blank. Solvent conditions, other absorbing compounds, and light scattering can affect the reading. Treat the sequence result as an estimate for the chosen model and cysteine state.

• Pace et al. (1995)
• ResidueBench extinction coefficient method