Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data

Institute of Molecular Biology, University of Oregon, Eugene 97403.
Analytical Biochemistry (Impact Factor: 2.22). 12/1989; 182(2):319-26. DOI: 10.1016/0003-2697(89)90602-7
Source: PubMed


Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.

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Article: Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data

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    • "Manipulations of DNA and E. coli were carried out using standard protocols [Sambrook et al., 1989]. Protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantified with the Bradford assay [Bradford, 1976] or estimated spectrophotometrically using the theoretical sequence-based coefficient of 33,265 M À1 cm À1 for SsMTAPII and 33,015 M À1 cm À1 for SsMTAP calculated at 280 nm for the hexameric protein [Gill and von Hippel, 1989]. SDS-PAGE was carried out as described by Weber et al. [1972]. "
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