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

• Published in: Analyst (London) 2014-03, Vol.139 (6), p.1442-1448
• Main Authors: Gómez-Mingot, Maria, Montiel, Vicente, Banks, Craig E, Iniesta, Jesús
• Format: Article
• Language: English
• Subjects: Animals; Biosensing Techniques; Cytochromes; Cytochromes c - chemistry; Cytochromes c - metabolism; Diffusion; Electrochemical Techniques; Electron transfer; Electron Transport; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Graphite; Graphite - chemistry; Horses; Hydrogen Peroxide - analysis; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; Immobilization; Media; Models, Molecular; Peroxidases - metabolism; Protein Conformation; Proteins
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c3an02137h

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 H 2 O 2 . Apparent Michaelis-Menten constant values ( K m ) 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. Electrochemical techniques have been used to study the pH-dependent dynamics of the direct charge-transfer processes of cytochrome c and the peroxidase activity after immobilization using SPGEs in a very wide range of pH values.