Extinction Coeffient

UV spectrophotometry is a useful tool for determining protein concentration in a solution. In order to take advantage of this method one needs an accurate measure of the protein of interest’s extinction coefficient (molar absorbtion coefficient). Assays that have been used include dry weight calculations, spectral methods, and colorimic techniques (Bradford and Lowry assays). These methods work well but require a fairly large amount of protein, yet modern protein sequencing methods allow us to use sequence data to determine extinction coefficients.

Assumptions:

The primary assumption is the spectral contributions of the tyrosine, tryptophan and cystine at 280 nm do not differ significantly in the native form of the protein, relative to the denatured form. The calculation is based on the Edlehoch model in which proteins are examined in a 6M guanidinium hydrochloride (Gdn-HCl) denaturing solution which allowed for matching of native to denatured forms. Another assumption is that the protein contains no other chromophores (thus those proteins containing prothetic groups absorbing UV and visible portions of the spectrum can’t be measured using this calculation).

The calculations is as follows:

E_M,Gdn-HCl=aE_M,Tyr + bE_M,Trp + cE_M,Cys

Where a,b,c are the number of tyrosine, trytophan and cystine residues per mole of protein and E_residue are the molar extinction rated of the residue at the wavelength used (280 nm). To get the extinction coefficient of the native protein Beer’s law is used:

Abs_Gdn-HCl/E_{M,Gdn-HCl= CDen}

which is equivalent to that of the native protein

Abs_nat/E_nat= C_nat

since the experimental conditions set the concentrations of both the denature and native form equal. Thus combining the equations above allows one to obtain the extinction coefficient of the native form:

(Abs_nat)(E_M,Gdn-HCl)/(Abs_M,Gdn-HCl).

Reference: Gill and von Hipple Anal Biochem 182,319-326 (1989)

Extinction Coefficients at 280nm

In 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer, pH 6.5.

Reference: Gill and von Hipple Anal Biochem 182,319-326 (1989)