Cytochrome c Mutants for Superoxide Biosensors
The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection o...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2009-04, Vol.81 (8), p.2976-2984 |
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| creator | Wegerich, Franziska Turano, Paola Allegrozzi, Marco Möhwald, Helmuth Lisdat, Fred |
| description | The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV−vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability. |
| doi_str_mv | 10.1021/ac802571h |
| format | Article |
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The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV−vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/ac802571h</identifier><identifier>PMID: 19296689</identifier><identifier>CODEN: ANCHAM</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Analytical chemistry ; Biochemistry ; Biological and medical sciences ; Biosensing Techniques - methods ; Biosensors ; Biotechnology ; Chemistry ; Circular Dichroism ; Cytochromes c - chemistry ; Cytochromes c - genetics ; Cytochromes c - metabolism ; Electrochemical methods ; Electrochemistry ; Electrodes ; Exact sciences and technology ; Fundamental and applied biological sciences. Psychology ; Heme - metabolism ; Humans ; Iron - metabolism ; Kinetics ; Magnetic Resonance Spectroscopy ; Methods. Procedures. Technologies ; Models, Molecular ; Mutant Proteins - chemistry ; Mutant Proteins - genetics ; Mutant Proteins - metabolism ; Mutation ; Protein Conformation ; Protein Folding ; Proteins ; Spectrometric and optical methods ; Spectrophotometry, Ultraviolet ; Spectrum analysis ; Structure-Activity Relationship ; Superoxides - analysis ; Superoxides - metabolism ; Various methods and equipments</subject><ispartof>Analytical chemistry (Washington), 2009-04, Vol.81 (8), p.2976-2984</ispartof><rights>Copyright © 2009 American Chemical Society</rights><rights>2009 INIST-CNRS</rights><rights>Copyright American Chemical Society Apr 15, 2009</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a436t-ef1cf7e76afeb1bb3b1f2deb6cf7361a7491a42da4a784c9cef644df021307053</citedby><cites>FETCH-LOGICAL-a436t-ef1cf7e76afeb1bb3b1f2deb6cf7361a7491a42da4a784c9cef644df021307053</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/ac802571h$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/ac802571h$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21397866$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19296689$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wegerich, Franziska</creatorcontrib><creatorcontrib>Turano, Paola</creatorcontrib><creatorcontrib>Allegrozzi, Marco</creatorcontrib><creatorcontrib>Möhwald, Helmuth</creatorcontrib><creatorcontrib>Lisdat, Fred</creatorcontrib><title>Cytochrome c Mutants for Superoxide Biosensors</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><description>The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV−vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.</description><subject>Analytical chemistry</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biosensing Techniques - methods</subject><subject>Biosensors</subject><subject>Biotechnology</subject><subject>Chemistry</subject><subject>Circular Dichroism</subject><subject>Cytochromes c - chemistry</subject><subject>Cytochromes c - genetics</subject><subject>Cytochromes c - metabolism</subject><subject>Electrochemical methods</subject><subject>Electrochemistry</subject><subject>Electrodes</subject><subject>Exact sciences and technology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Heme - metabolism</subject><subject>Humans</subject><subject>Iron - metabolism</subject><subject>Kinetics</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Methods. Procedures. Technologies</subject><subject>Models, Molecular</subject><subject>Mutant Proteins - chemistry</subject><subject>Mutant Proteins - genetics</subject><subject>Mutant Proteins - metabolism</subject><subject>Mutation</subject><subject>Protein Conformation</subject><subject>Protein Folding</subject><subject>Proteins</subject><subject>Spectrometric and optical methods</subject><subject>Spectrophotometry, Ultraviolet</subject><subject>Spectrum analysis</subject><subject>Structure-Activity Relationship</subject><subject>Superoxides - analysis</subject><subject>Superoxides - metabolism</subject><subject>Various methods and equipments</subject><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpl0M9LwzAUB_AgipvTg_-AFMGDh873kixpj1r8BRMP6rmkacI6tmYmLbj_3sjKdvD04PHh_fgScokwRaB4p3QGdCZxcUTGOKOQiiyjx2QMACylEmBEzkJYAiACilMywpzmQmT5mEyLbef0wru1SXTy1neq7UJinU8--o3x7qepTfLQuGDa4Hw4JydWrYK5GOqEfD09fhYv6fz9-bW4n6eKM9GlxqK20kihrKmwqliFltamErHLBCrJc1Sc1oormXGda2MF57WNzzCQMGMTcr2bu_HuuzehK5eu921cWVKUOaAEFtHtDmnvQvDGlhvfrJXflgjlXzDlPphor4aBfbU29UEOSURwMwAVtFpZr1rdhL2Lh-UyE-LglA6Ho_4v_AWFinWk</recordid><startdate>20090415</startdate><enddate>20090415</enddate><creator>Wegerich, Franziska</creator><creator>Turano, Paola</creator><creator>Allegrozzi, Marco</creator><creator>Möhwald, Helmuth</creator><creator>Lisdat, Fred</creator><general>American Chemical Society</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7TA</scope><scope>7TB</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope></search><sort><creationdate>20090415</creationdate><title>Cytochrome c Mutants for Superoxide Biosensors</title><author>Wegerich, Franziska ; Turano, Paola ; Allegrozzi, Marco ; Möhwald, Helmuth ; Lisdat, Fred</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a436t-ef1cf7e76afeb1bb3b1f2deb6cf7361a7491a42da4a784c9cef644df021307053</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Analytical chemistry</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biosensing Techniques - methods</topic><topic>Biosensors</topic><topic>Biotechnology</topic><topic>Chemistry</topic><topic>Circular Dichroism</topic><topic>Cytochromes c - chemistry</topic><topic>Cytochromes c - genetics</topic><topic>Cytochromes c - metabolism</topic><topic>Electrochemical methods</topic><topic>Electrochemistry</topic><topic>Electrodes</topic><topic>Exact sciences and technology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Heme - metabolism</topic><topic>Humans</topic><topic>Iron - metabolism</topic><topic>Kinetics</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Methods. Procedures. Technologies</topic><topic>Models, Molecular</topic><topic>Mutant Proteins - chemistry</topic><topic>Mutant Proteins - genetics</topic><topic>Mutant Proteins - metabolism</topic><topic>Mutation</topic><topic>Protein Conformation</topic><topic>Protein Folding</topic><topic>Proteins</topic><topic>Spectrometric and optical methods</topic><topic>Spectrophotometry, Ultraviolet</topic><topic>Spectrum analysis</topic><topic>Structure-Activity Relationship</topic><topic>Superoxides - analysis</topic><topic>Superoxides - metabolism</topic><topic>Various methods and equipments</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wegerich, Franziska</creatorcontrib><creatorcontrib>Turano, Paola</creatorcontrib><creatorcontrib>Allegrozzi, Marco</creatorcontrib><creatorcontrib>Möhwald, Helmuth</creatorcontrib><creatorcontrib>Lisdat, Fred</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wegerich, Franziska</au><au>Turano, Paola</au><au>Allegrozzi, Marco</au><au>Möhwald, Helmuth</au><au>Lisdat, Fred</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cytochrome c Mutants for Superoxide Biosensors</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2009-04-15</date><risdate>2009</risdate><volume>81</volume><issue>8</issue><spage>2976</spage><epage>2984</epage><pages>2976-2984</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><abstract>The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV−vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>19296689</pmid><doi>10.1021/ac802571h</doi><tpages>9</tpages></addata></record> |
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| subjects | Analytical chemistry Biochemistry Biological and medical sciences Biosensing Techniques - methods Biosensors Biotechnology Chemistry Circular Dichroism Cytochromes c - chemistry Cytochromes c - genetics Cytochromes c - metabolism Electrochemical methods Electrochemistry Electrodes Exact sciences and technology Fundamental and applied biological sciences. Psychology Heme - metabolism Humans Iron - metabolism Kinetics Magnetic Resonance Spectroscopy Methods. Procedures. Technologies Models, Molecular Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - metabolism Mutation Protein Conformation Protein Folding Proteins Spectrometric and optical methods Spectrophotometry, Ultraviolet Spectrum analysis Structure-Activity Relationship Superoxides - analysis Superoxides - metabolism Various methods and equipments |
| title | Cytochrome c Mutants for Superoxide Biosensors |
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