Protein Extinction Coefficient & MW from Sequence

Understanding the protein extinction coefficient and molecular weight calculator — concept and principle

Before you can measure a protein's concentration by A280, you need its molar extinction coefficient ε, and to convert molarity to mg/mL you need its molecular weight. This calculator predicts both from the amino-acid sequence in one-letter code. It counts tryptophan, tyrosine and cysteine residues, applies the Pace et al. (1995) coefficients, and sums average residue masses for the molecular weight.

How it is calculated

• ε280 (reduced Cys) = 5,500 × nW + 1,490 × nY (M⁻¹cm⁻¹).
• ε280 (all Cys as cystines) = 5,500 × nW + 1,490 × nY + 125 × ⌊nC ÷ 2⌋, since each disulfide bond (cystine) uses two cysteines.
• Average MW = Σ residue masses + 18.015 Da, the extra water accounting for the free N- and C-termini.
• Length: number of residues counted.

The principle

At 280 nm, the indole ring of tryptophan absorbs strongly, the phenol ring of tyrosine absorbs about a quarter as much, and a disulfide bond absorbs weakly. Other residues hardly absorb. Pace and colleagues measured many proteins of known concentration and found that adding up these three contributions, with values for folded proteins in water, predicts ε well. Residue masses are amino-acid masses minus one water each, because a water molecule is released when each peptide bond forms; one water is then added back for the whole chain.

Worked example

1. Sequence: MKWVTFISLLLLFSSAYS (18 residues; the signal peptide of the bovine serum albumin precursor).
2. Counts: W = 1, Y = 1, C = 0.
3. ε280 = 5,500 × 1 + 1,490 × 1 = 6,990 M⁻¹cm⁻¹; with no cysteines, the cystine value is also 6,990.
4. Residue masses: M 131.19 + K 128.17 + W 186.21 + V 99.13 + T 101.11 + F 2 × 147.18 + I 113.16 + S 4 × 87.08 + L 4 × 113.16 + A 71.08 + Y 163.18 = 2,088.54 Da.
5. MW = 2,088.54 + 18.02 = 2,106.55 Da; length = 18.

How to read the result

Use the cystine value for proteins that form disulfides and the reduced value for intracellular proteins or samples in DTT or TCEP. A protein with no Trp or Tyr has ε near zero at 280 nm, and A280 cannot be used.

Common mistakes

• Pasting a sequence that includes the signal peptide or tag when the purified protein lacks it.
• Using the cystine value for a protein that has free cysteines.
• Assuming the predicted MW includes glycosylation or other modifications; it does not.

Frequently asked questions

Why use average rather than monoisotopic mass?

Average mass suits concentration work with whole molecules. Mass spectrometry often needs monoisotopic mass.

Does the denatured protein have the same ε?

Not exactly; unfolding shifts aromatic absorption slightly. Gill and von Hippel described a method based on denatured protein.

Is 125 per cystine a large effect?

Usually small, but it matters in proteins with few aromatics and many disulfides.

Sources: C. N. Pace, F. Vajdos, L. Fee, G. Grimsley and T. Gray, Protein Science 4, 2411–2423 (1995) · S. C. Gill and P. H. von Hippel, Analytical Biochemistry 182, 319–326 (1989)