How to measure and predict the molar absorption coefficient of a protein

TLDR

The molar absorption coefficient of a protein is typically determined from concentration estimates based on dry weight, nitrogen, or amino acid analysis, and its 280‑nm absorbance depends on tryptophan, tyrosine, and cystine content, as described by Gill and von Hippel. The study aims to determine the most reliable approach for measuring protein molar absorption coefficients and finds that the Edelhoch method is preferable to prediction. The authors estimated average ε values for tryptophan, tyrosine, and cystine in 18 proteins and measured these values in water, propanol, 6 M guanidine hydrochloride, and 8 M urea. The study shows that the Edelhoch method is the most accurate way to measure ε, while a predictive equation ϵ(280) = 5,500 × #Trp + 1,490 × #Tyr + 125 × #cystine provides reliable estimates for proteins containing tryptophan but is less accurate for proteins lacking Trp.

Abstract

The molar absorption coefficient, ε, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring ε for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319–326] and is based on data from Edelhoch [1967, Biochemistry 6:1948–1954]).) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average ε values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the ε values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average ε values for the proteins are less than the ε values measured in any of the solvents. For Tyr, the average ε values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured ε values for 80 proteins, the ε at 280 nm of a folded protein in water, ε(280), can best be predicted with this equation ϵ(280) (M−1 cm−1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125). These ε(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict ε.