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Protein Extinction Coefficients and Concentration Calculation

Stanley C. Gill and Peter H. von Hippel presented a method for calculation accurate(to ±5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition3.

This is an online tool for protein extinction coefficient and concentration calculation. Please input the amino acid sequence of your protein in the text-box below.

1. Sequence (Paste the raw sequence, not fasta format):

Full length:0

2. Absorbance at 280 nm(A280nm):



Absorbance and Extinction Coefficients

Beer’s Law states that molar absorptivity is constant (and the absorbance is proportional to concentration) for a given substance dissolved in a given solute and measured at a given wavelength. For this reason, molar absorptivities are called molar absorption coefficients or molar extinction coefficients. Standard laboratory spectrophotometers are fitted for use with 1cm-width sample cuvettes; hence, the path length is generally assumed to be equal to one and the term is dropped altogether in most calculations.

Aλ = εcL = εc, when L = 1cm and where c is the protein concentration.

The molar absorption coefficient of a peptide or protein is related to its tryptophan (W), tyrosine (Y) and cysteine (C) amino acid composition. At 280nm, this value is approximated by the weighted sum of the 280nm molar absorption coefficients of these three constituent amino acids, as described in the following equation:

εmolar = (nW * 5500) + (nY * 1490) + (nC * 125)

where n is the number of each residue and the stated values are the amino acid molar absorptivities at 280nm and εmolar is molar extinction coefficient.

Solving the expression of Beer’s law for concentration, one can easily see what values are needed to determine the concentration of a peptide or protein solution:

c = A / εL, when L=1cm c = A / ε.

If one wishes to report concentration in terms of mg/ml, then an adjustment factor of 10 must be made when using these percent solution extinction coefficients (i.e., one must convert from 10 mg/ml units to 1 mg/ml concentration units).

(A / εpercent) 10 = concentration in mg/ml

The relationship between molar extinction coefficient (εmolar) and percent extinction coefficient (εpercent) is as follows:

molar) × 10 = (εpercent) × (molecular weight of protein)

Thus, A×(molecular weight of the protein)/εmolar=concentration in mg/ml

Most mammalian antibodies (i.e., immunoglobulins) have protein extinction coefficients around 210000M-1cm-1. For a typical IgG with MW = 150,000, the concentration could be calculated as A/1.4 mg/ml and for BSA, it is A/0.66 mg/ml (MW=66400, εmolar=43824M-1cm-1)

References

  • 1. Lange’s Handbook of Chemistry, 14th Edition, Dean, J.A., Ed. (1992). McGraw-Hill, Inc., New York.
  • 2. Handbook of Chemistry and Physics, 56th Edition, Weast, R.C., Ed. (1975). CRC Press, Cleveland.
  • 3. Gill, S.C. and von Hippel, P.H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-26.
  • 4. Pace, C.N., et al. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411-23.
  • 5. Practical Handbook of Biochemistry and Molecular Biology, Fasman, D.G., Ed. (1992). CRC Press, Boston.