An indication how much light a protein is absorbing at a certain wavelength can be derived from the extinction coefficient. It is helpful to have an estimation of this coefficient to be able to follow a protein within a spectrophotometer. The knowledge of the amino acid composition is sufficient for an estimation of the molar extinction coefficient of a protein [1]. The extinction coefficient of the native protein in water can be calculated with the following equation which includes the molar extinction coefficient of tyrosine, tryptophan and cystine (consider cystine for calculation due to cysteine's property to not absorb appreciably at wavelengths >260 nm) for a given wavelength:
E(Prot) = Number(Tyr) * Ext(Tyr) + Number(Trp) * Ext(Trp) + Number(Cystine) * Ext(Cystine)
where (for proteins in water measured at 280 nm):
Ext(Tyr) = 1490, Ext(Trp) = 5500, Ext(Cystine) = 125;
We are using the Edelhoch method for the calculation [2], but with different extinction coefficients for Trp and Tyr which were determined by Pace et al.[3]. Edelhoch determined the values for extinction coefficients for Trp and Tyr in pH 6.5, 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer. Extinction coefficients are in units of M-1 * cm-1, at 280 nm measured in water.
Note: Cystin is an amino acid dimer and consists of two cysteine molecules which are joined by a disulfide bond.
The first two calculations (extinction coefficient and absorbance [optical density]) show the computed value based on the assumption that all cysteine residues appear as half cystines. The second two calculations assume that no cystine formation was build (no cystein appears as half cystine). Experience shows that the computation is quite reliable for proteins containing Trp residues, however there may be more than 10% error for proteins without Trp residues. This is being indicated above the calculations if this case occurs.
[1] Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319-326.
[2] Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954.
[3] Pace CN, Vajdos F, Fee L, Grimsley G, Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 11, 2411-2423.