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Protein Extinction Coefficient Calculator

Calculate a protein's extinction coefficient at 280 nm from its Trp, Tyr and cystine content, with the absorbance of 1 mg/mL and the concentration from A280.

Formula

ε280=5500 nTrp+1490 nTyr+125 ncystine\varepsilon_{280} = 5500\,n_{\mathrm{Trp}} + 1490\,n_{\mathrm{Tyr}} + 125\,n_{\mathrm{cystine}}
A0.1%=ε280MWA^{0.1\%} = \dfrac{\varepsilon_{280}}{\mathrm{MW}}
c (mg/mL)=A280A0.1% ℓc\,(\mathrm{mg/mL}) = \dfrac{A_{280}}{A^{0.1\%}\,\ell}
nTrp, nTyrn_{\mathrm{Trp}},\ n_{\mathrm{Tyr}}
numbers of tryptophan and tyrosine residues
ncystinen_{\mathrm{cystine}}
number of disulfide-bonded cysteine pairs
A0.1%A^{0.1\%}
absorbance of a 1 mg/mL solution at a 1 cm path

How it works

In the near ultraviolet a protein absorbs mainly through its tryptophan and tyrosine side chains, and to a small extent through disulfide bonds. Pace and colleagues measured the contributions and fitted the values 5500, 1490 and 125 M⁻¹ cm⁻¹ per residue. Adding them up gives the molar extinction coefficient, and dividing by the molecular weight gives the absorbance of 1 mg/mL.

With that in hand, a reading of A280 converts to a concentration. The cysteine treatment matters only a little: free cysteines do not absorb, while each disulfide adds 125. The tool offers both assumptions. A protein with none of the three absorbing groups cannot be measured at 280 nm at all.

Worked example

Hen egg-white lysozyme (129 residues): 6 tryptophan, 3 tyrosine, 8 cysteine (4 disulfide bonds).

  1. ε₂₈₀ = 5500 × 6 + 1490 × 3 + 125 × 4 = 33,000 + 4,470 + 500 = 37,970 M⁻¹ cm⁻¹.
  2. MW = 14,313 g/mol, so A 0.1% = 37,970 / 14,313 = 2.65.

ε₂₈₀ = 37,970 M⁻¹ cm⁻¹ and A 0.1% = 2.65, in line with the values commonly quoted for lysozyme.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The native, folded protein in aqueous buffer near neutral pH.
  • Each Trp, Tyr and cystine contributes independently with the average values above.
  • No cofactors, prosthetic groups or modifications that absorb near 280 nm.

Common mistakes

  • Using a calculated value when the protein binds a chromophore such as heme or a flavin. Their absorbance must be measured.
  • Measuring a turbid or aggregated sample, where light scattering adds to the apparent absorbance.
  • Applying the coefficient in buffers with high absorbance at 280 nm, or in the presence of nucleic acid.

Related tools

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.