Engineered Superoxide Dismutase Monomers for Superoxide Biosensor Applications

Engineered Superoxide Dismutase Monomers for Superoxide Biosensor Applications

Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional c...

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Bibliographic Details
Published in:Analytical chemistry (Washington) Vol. 78; no. 3; pp. 928 - 935
Main Authors: Beissenhirtz, Moritz K, Scheller, Frieder W, Viezzoli, Maria S, Lisdat, Fred
Format: Journal Article
Language:English
Published: Washington, DC American Chemical Society 01-02-2006
Subjects:
Analytical chemistry
Biological and medical sciences
Biomedical research
Biosensing Techniques - instrumentation
Biosensing Techniques - methods
Biosensors
Biotechnology
Chemistry
Electrochemical methods
Electrochemistry
Electrodes
Exact sciences and technology
Fundamental and applied biological sciences. Psychology
General, instrumentation
Humans
Methods. Procedures. Technologies
Protein Engineering
Sensitivity and Specificity
Superoxide Dismutase - analysis
Various methods and equipments
Human
Gold
Cysteine
Enzyme
Electrochemical method
Interference
Immobilization
Electron transfer
Chemical sensor
Reaction rate
Protein
Cytochrome c
Sensitivity
Residue
Biosensor
Thermodynamic parameter
Amperometry
Oxidation
Superoxide radicals
Kinetic parameter
Sulfur
Quantitative analysis
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Summary:Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasi-reversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD−mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by ∼1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold.
Bibliography:istex:D707EB2F42FF8E27C7D40823438389592CCF5F8D
ark:/67375/TPS-3Q6XC7G1-7
ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0003-2700
1520-6882
DOI:10.1021/ac051465g