Engineered Superoxide Dismutase Monomers for Superoxide Biosensor Applications

Abstract

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 approximately 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.

Electrochemical and Sensorial Behavior of SOD Mutants Immobilized on Gold Electrodes in Aqueous/Organic Solvent Mixtures

“…As shown previously cysteine mutants of a monomeric form of the human Cu,Zn-SOD can be prepared and immobilized on gold electrodes via the thiol group introduced [24]. For the investigation of the influence of an organic solvent on the electrochemical behavior of the fixed enzyme a mutant with two mutation sites was chosen.…”

“…The human Cu,Zn-SOD mutant was prepared as described previously [24]. Starting from a monomeric, human version of the enzyme, Glycine 61 and Serine 142 were replaced by cysteine residues.…”

“…The SOD mutant electrode can be successfully applied for superoxide detection in aqueous media [24]. Two sensor modes are feasible: One is based on the reductase activity of the dismutase when the electrode is poised at negative potential (with respect to E f ) and a reduction current can be detected in the presence of superoxide.…”

“…Recently cysteine mutants of a monomeric Cu,Zn-SOD have been prepared and used for the direct immobilization of the protein on a gold electrode avoiding interferences from the intermittent promoter layer. These electrodes were also suitable for a sensitive radical measurement [24]. So far, however, these electrodes have not been applied in mixtures of aqueous buffer and organic solvent.…”

Recognition of Errors in the Refinement and Validation of Three-Dimensional Structures of AC1 Proteins of Begomovirus Strains by Using ProSA-Web

“…The analysis of protein structures is generally 2 Journal of Viruses [13][14][15], theoretical models [16][17][18][19], and protein engineering [20,21]. For in silico ligand designing to be an effective inhibitor, Rep protein of selected begomovirus strains (YP 003288785, YP 002004579, and YP 003288773), which is responsible for replication, was used.…”