What absorbs at 280 nanometres, and what does not
Almost all of a peptide's absorbance at 280 nm comes from tryptophan, with a smaller contribution from tyrosine and a small one from cystine, the oxidised disulfide form of cysteine. Phenylalanine absorbs further into the ultraviolet and contributes essentially nothing at 280.
The consequence is blunt. A peptide with no tryptophan and no tyrosine has an extinction coefficient of zero at 280 nm, and its concentration cannot be determined by absorbance at that wavelength however carefully the measurement is made. Many short research peptides fall into this category, BPC-157 and GHK among them.
What to do when the coefficient is zero
Measuring at 205 or 214 nanometres works, because the peptide bond itself absorbs there. The trade-off is that almost everything else absorbs there too, so the buffer has to be transparent at that wavelength and even small amounts of solvent or scavenger interfere.
Colorimetric assays such as BCA or Bradford are the other route. They measure total peptide against a standard curve and are indifferent to aromatic content, at the cost of requiring a standard and a reaction step.
Native and denatured coefficients
The Pace method gives two figures. The native, oxidised value counts 125 per disulfide bridge along with the tryptophan and tyrosine contributions. The denatured, reduced value drops the disulfide term entirely, because a reduced sample has no cystine in it.
Which one applies depends on the sample, not on the peptide. A sample containing DTT or TCEP reads the reduced value. Using the wrong one on a peptide with several bridges introduces an error of a few hundred M⁻¹cm⁻¹, which matters when the tryptophan count is low and the bridges are a large fraction of the total.
Absorptivity, and why it is the more convenient number
Molar extinction coefficient relates absorbance to molar concentration. Dividing it by the molecular weight gives absorptivity, which relates absorbance directly to milligrams per millilitre. That is the form quoted as the A 0.1 percent value or as the absorbance of a 1 mg/mL solution.
For a typical protein the absorptivity is close to 1, which is where the useful shorthand comes from that an A280 of 1 is about 1 mg/mL. For peptides it varies enormously, because a small peptide with one tryptophan has a far higher absorptivity than a large protein with the same one tryptophan.
How the extinction coefficient is calculated
The Pace method: a weighted count of the three absorbing species. It is a summation, not a fit, so the result is exact for the composition given and approximate for the real molecule.
epsilon(280) = 5500 x nTrp + 1490 x nTyr + 125 x nCystine absorptivity = epsilon / molecular weight [ (mg/mL)^-1 cm^-1 ] concentration = A / (epsilon x path length) [ mol/L ]
- Count the chromophores. Tryptophan, tyrosine and cysteine, from the parsed sequence. Cysteines are paired into cystines by default, and the count can be overridden if the real number of bridges is known.
- Apply the Pace coefficients. 5,500 per tryptophan, 1,490 per tyrosine, 125 per cystine bridge. These are the values from the 1995 paper and are the same ones ProtParam uses.
- Drop the cystine term for reduced samples. Selecting denatured conditions removes the 125 per bridge, giving the figure that applies to a sample in a reducing agent.
- Divide by molecular weight for absorptivity. Gives the absorbance of a 1 mg/mL solution in a 1 cm cell, which is the more convenient form when working in mass rather than molar terms.
- Invert Beer-Lambert for concentration. Absorbance over the product of coefficient and path length gives molar concentration, and multiplying that by molecular weight gives milligrams per millilitre directly, since mol/L times g/mol is g/L.
What this method cannot tell you
- •The Pace values were derived for folded proteins in water. Local environment shifts real coefficients by a few percent, and more in unusual solvents.
- •It assumes cysteines are fully paired or fully reduced. A partially oxidised sample sits between the two figures.
- •It says nothing about purity. A contaminant that absorbs at 280 nm is counted as if it were peptide.
- •Nucleic acid contamination absorbs strongly at 260 nm and substantially at 280. A 260 over 280 ratio well above 0.6 indicates the reading is not measuring only peptide.
Where the numbers come from
Extinction coefficient calculator: frequently asked questions
A measure of how strongly a substance absorbs light at a given wavelength, in units of inverse molar inverse centimetre. It is the proportionality constant in the Beer-Lambert law.
A coefficient of 5,500 M⁻¹cm⁻¹ means a 1 molar solution in a 1 cm cell would give an absorbance of 5,500, which is why real measurements are made on much more dilute solutions.
Tryptophan and tyrosine, with a minor contribution from cystine, the disulfide-bridged form of cysteine.
- •Tryptophan: 5,500 M⁻¹cm⁻¹
- •Tyrosine: 1,490 M⁻¹cm⁻¹
- •Cystine bridge: 125 M⁻¹cm⁻¹
Phenylalanine absorbs at shorter wavelengths and contributes essentially nothing at 280 nm.
Then its extinction coefficient at 280 nm is zero or nearly so, and absorbance at that wavelength cannot measure its concentration.
The alternatives are measuring at 205 or 214 nm, where the peptide bond itself absorbs, or a colorimetric assay such as BCA. Both have their own constraints, chiefly buffer interference for the low-wavelength route and the need for a standard curve for the colorimetric one.
A published procedure for predicting a protein's 280 nm extinction coefficient from its amino acid composition, using coefficients fitted to a set of proteins with measured values.
It predicts measured coefficients to within a few percent for most sequences, which is why it is the standard approach and the one ProtParam implements.
Because the 125 per disulfide bridge only applies when bridges are present. A reduced sample has none.
The choice depends on the sample, not the peptide. A solution containing DTT or TCEP reads the reduced figure.
Divide the absorbance by the product of the extinction coefficient and the path length. That gives moles per litre.
To reach milligrams per millilitre, multiply the molar figure by the molecular weight. Moles per litre times grams per mole is grams per litre, and grams per litre and milligrams per millilitre are the same number.
Between about 0.1 and 1.0. Below 0.1 the signal is close to the noise floor; above about 1.5 most instruments leave their linear range.
A reading outside that window should be brought back into it by diluting the sample or changing the cuvette path length, not by trusting the number.
The extinction coefficient divided by the molecular weight: the absorbance of a 1 mg/mL solution in a 1 cm cell.
It is the convenient form when you think in mass rather than molar terms. Typical proteins land near 1, which is the origin of the rule of thumb that an A280 of 1 is roughly 1 mg/mL. Peptides vary far more widely than that.
Absorbance is directly proportional to it. A 0.5 cm cell gives half the reading of a 1 cm cell at the same concentration.
Microvolume spectrophotometers use very short path lengths, often 1 mm or less, and either report a normalised figure or require you to enter the real path length. Getting this wrong is a tenfold error.
Whether nucleic acid is contaminating the sample. Pure protein sits around 0.5 to 0.6; pure DNA is about 1.8.
A ratio well above 0.6 means the 280 nm reading includes absorbance from something that is not peptide, and the calculated concentration is too high.
Most common buffers are transparent at 280 nm. Blanking against the buffer removes any residual contribution.
At 205 or 214 nm the picture is very different. Chloride, acetate, TFA and DMSO all absorb strongly there, which is the main practical obstacle to using the low-wavelength route.
Usually within a few percent of a measured value for a folded protein in water. Short peptides and unusual solvents widen the gap.
The coefficients were fitted to folded proteins, and a chromophore's environment affects its absorbance. A tryptophan fully exposed to solvent in a short peptide is not in the same environment as one buried in a protein core.
At 280 nm, yes: DMSO is reasonably transparent there. At lower wavelengths it absorbs strongly and rules the measurement out.
Blank against the same DMSO concentration, since the co-solvent also changes the refractive index and the baseline.
Because one entry claimed a coefficient for a peptide that has no chromophore at all, and another was labelled a fragment when it was a full-length protein.
Every preset now carries the coefficient computed from its own sequence, and the ones with no absorbance at 280 nm are labelled as such rather than being given a borrowed number.
Mildly. Tyrosine's absorbance shifts noticeably above about pH 10, where its phenol group deprotonates.
In the neutral range where most work is done, the effect is small enough to ignore. At high pH it is not.
Yes, and this is what the Pace method was built for. It is more reliable for a folded protein than for a short peptide.
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