Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity.
The direct electron transfer of cytochrome c has been studied at screen-printed graphite macroelectrodes without recourse to mediators or the need for any electrode pre-treatment as is commonly employed within the literature. A wide range of pH values from 2.0 to 11.0 have been explored upon the electrochemical response of cytochrome c and different voltammetric signatures have been observed. The direct electron transfer of the alkaline transition of cytochrome c was found impeded within alkaline media leading to either an irreversible redox process or even no voltammetric responses. In acidic aqueous media the electrochemical process is observed to undergo a mixed diffusion and adsorption controlled process rather than a purely diffusional process of the native conformation as observed at pH 7.0. Interestingly, at pH 3.5 a new conformational state is revealed in cooperation with the native conformation. The immobilization of the protein was satisfactorily obtained using a simple method by cycling the protein at specific solution pH values allowing amperometric responses to be obtained and gives rise to useful pseudo-peroxidase activity for sensing H2O2. Apparent Michaelis-Menten constant values (Km) were calculated via the Lineweaver-Burk method with deduced values of 25 ± 4, 98 ± 12 and 230 ± 30 mM, respectively for pH values of 2.0, 3.0 and 7.0. Such work is important for those utilising cytochrome c in bio-electrochemical and related applications.
Physical Chemistry Department and Institute of Electrochemistry, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain. jesus.iniesta@ua.es.
Maria
M
Gómez-Mingot
Vicente
V
Montiel
Craig E
CE
Banks
Jesús
J
Iniesta
D000818:Animals; D015374:Biosensing Techniques; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D055664:Electrochemical Techniques; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006108:Graphite / Q000737:chemistry*; D006736:Horses; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation
D004800:Enzymes, Immobilized; D006108:Graphite; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D010544:Peroxidases