Binding Stoichiometry of DNA Adducts with Antibody Studied by Capillary Electrophoresis and Laser-Induced Fluorescence
Identifieur interne : 000D26 ( Istex/Checkpoint ); précédent : 000D25; suivant : 000D27Binding Stoichiometry of DNA Adducts with Antibody Studied by Capillary Electrophoresis and Laser-Induced Fluorescence
Auteurs : Hailin Wang [Canada] ; James Xing [Canada] ; Woei Tan [Canada] ; Mike Lam [Canada] ; Trevor Carnelley [Canada] ; Michael Weinfeld [Canada] ; X. Chris Le [Canada]Source :
- Analytical Chemistry [ 0003-2700 ] ; 2002.
Abstract
Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE−16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE−16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE−16-mer and an unlabeled BPDE−90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR−BPDE−16-mer molecule, antibody with two TMR−BPDE−16-mer molecules, and antibody with one TMR−BPDE−16-mer and one BPDE−90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.
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DOI: 10.1021/ac0201979
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Antibody Studied by Capillary Electrophoresis and
Laser-Induced Fluorescence</title><author><name sortKey="Wang, Hailin" sort="Wang, Hailin" uniqKey="Wang H" first="Hailin" last="Wang">Hailin Wang</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation></author><author><name sortKey="Xing, James" sort="Xing, James" uniqKey="Xing J" first="James" last="Xing">James Xing</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation></author><author><name sortKey="Tan, Woei" sort="Tan, Woei" uniqKey="Tan W" first="Woei" last="Tan">Woei Tan</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation></author><author><name sortKey="Lam, Mike" sort="Lam, Mike" uniqKey="Lam M" first="Mike" last="Lam">Mike Lam</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation></author><author><name sortKey="Carnelley, Trevor" sort="Carnelley, Trevor" uniqKey="Carnelley T" first="Trevor" last="Carnelley">Trevor Carnelley</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation></author><author><name sortKey="Weinfeld, Michael" sort="Weinfeld, Michael" uniqKey="Weinfeld M" first="Michael" last="Weinfeld">Michael Weinfeld</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation><affiliation></affiliation></author><author><name sortKey="Le, X Chris" sort="Le, X Chris" uniqKey="Le X" first="X. Chris" last="Le">X. Chris Le</name><affiliation wicri:level="1"><country xml:lang="fr">Canada</country><wicri:regionArea>Department of Public Health Sciences and Cross Cancer Institute, University of Alberta,Edmonton, Alberta T6G 2G3</wicri:regionArea><wicri:noRegion>Alberta T6G 2G3</wicri:noRegion></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Analytical Chemistry</title><title level="j" type="abbrev">Anal. Chem.</title><idno type="ISSN">0003-2700</idno><idno type="eISSN">1520-6882</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2002</date><biblScope unit="vol">74</biblScope><biblScope unit="issue">15</biblScope><biblScope unit="page" from="3714">3714</biblScope><biblScope unit="page" to="3719">3719</biblScope></imprint><idno type="ISSN">0003-2700</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0003-2700</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE−16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE−16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE−16-mer and an unlabeled BPDE−90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR−BPDE−16-mer molecule, antibody with two TMR−BPDE−16-mer molecules, and antibody with one TMR−BPDE−16-mer and one BPDE−90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.</div></front></TEI><affiliations><list><country><li>Canada</li></country></list><tree><country name="Canada"><noRegion><name sortKey="Wang, Hailin" sort="Wang, Hailin" uniqKey="Wang H" first="Hailin" last="Wang">Hailin Wang</name></noRegion><name sortKey="Carnelley, Trevor" sort="Carnelley, Trevor" uniqKey="Carnelley T" first="Trevor" last="Carnelley">Trevor Carnelley</name><name sortKey="Lam, Mike" sort="Lam, Mike" uniqKey="Lam M" first="Mike" last="Lam">Mike Lam</name><name sortKey="Le, X Chris" sort="Le, X Chris" uniqKey="Le X" first="X. Chris" last="Le">X. Chris Le</name><name sortKey="Tan, Woei" sort="Tan, Woei" uniqKey="Tan W" first="Woei" last="Tan">Woei Tan</name><name sortKey="Weinfeld, Michael" sort="Weinfeld, Michael" uniqKey="Weinfeld M" first="Michael" last="Weinfeld">Michael Weinfeld</name><name sortKey="Xing, James" sort="Xing, James" uniqKey="Xing J" first="James" last="Xing">James Xing</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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