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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    • "All histatin 5 concentrations were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., U.S.A.) at 280 nm. The extinction coefficient used was 2560 M À1 cm À1 , calculated from the tyrosine content of the peptide[22,23]. Refractive index measurements. The refractive indices of the buffers were measured using an Abbe 60 refractometer (Bellingham & Stanley Ltd., UK) at wavelengths of 579.1, 546.1 and 435.8 nm. "
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    ABSTRACT: Hypothesis: The adsorption of histatin 5 to hydrophilic silica surfaces is governed by electrostatic attractive forces between the positive protein and the negative surface. Hence pH and ionic strength control the adsorbed amount, which can be described by coarse-grained Monte Carlo simulations accounting for electrostatic forces and charge regulation of the protein. Experiments: The amount of histatin 5 adsorbed to hydrophilic silica surfaces at different pH and ionic strengths was measured using null ellipsometry. The results were compared with coarse-grained Monte Carlo simulations of a single histatin 5 molecule and a surface with a fixed, smeared charge set according to experimental values for silica. The Langmuir isotherm was used to calculate the surface coverage from the simulation results. The effect of charge regulation of the protein was investigated. Findings: Even though electrostatic attractive forces are important for the investigated system, a non-electrostatic short-ranged attraction with a strength of about 2.9kBT per amino acid was needed in the simulations to get surface coverages close to experimental values. The importance of electrostatics increases with increasing pH. Charge regulation of the protein affected the results from the simulations only at high surface charge and low ionic strength.
    Full-text · Article · Jan 2016 · Journal of Colloid and Interface Science
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    • "(Accession number: P0C0C7). These include the molecular weight, amino acid composition, theoretical isoelectric point (pI), extinction coefficient (Gill and von Hippel, 1989), and instability index (Guruprasad et al., 1990). All were computed using the ProtParam tool of ExPAsy server ( "

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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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    ABSTRACT: The combination of the gene of purine nucleoside phosphorylase (PNP) from E. coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with kcat/Km values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign. This article is protected by copyright. All rights reserved
    No preview · Article · Oct 2015 · Journal of Cellular Biochemistry
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