Thin electrochemically active films of cytochrome P450s, putidaredoxin, and hemoglobin

Date of Completion

January 1999

Keywords

Chemistry, Analytical|Chemistry, Biochemistry|Engineering, Biomedical|Environmental Sciences

Degree

Ph.D.

Abstract

The purpose of this research was to develop novel electrochemically active films consisting of various proteins for possible applications in biosensors, chemical toxicity detection, and carcinogenicity studies. ^ Two unique and potentially general methods were used to incorporate proteins into the films. In the first method, a cationic surfactant, didodecyldimethylammonium bromide (DDAB) or a zwitterionic surfactant mixture, lecithin was used to make stable ordered films containing hemoglobin (Hb), cytochrome c (cyt c) and alcohol dehydrogenase (ADH). The electron transfer rates of Hb and cyt c in films on electrodes were 2.3 s−1 and 0.3 s −1, greatly enhanced compared to those in solution. An underlying Nafion film on the PG electrodes improved the stability of the protein-surfactant films. Hb in films on electrodes was used for electrocatalytic reduction of trichloroacetic acid. Crosslinked Nafion-lecithin-alcohol dehydrogenase (ADH) films retained nearly 85% of enzyme activity after 2 days of storage at 22°C compared to 38% for ADH stored in buffer solutions at 4°C for 2 days. ^ The phase transition temperatures of Hb-DDAB and Nafion-DDAB-Hb films were estimated at 17°C and 16°C, respectively. Microstructural investigation suggested that Hb-DDAB and Nafion-Hb-DDAB films exist as islands on the PG surface. Furthermore, spectroscopic studies showed that redox protein molecules are oriented and not extensively denatured in the films. ^ The second method of film preparation involved proteins assembled onto gold electrodes via layer-by-layer growth with polyions of opposite charges. Reversible electrochemistry was achieved for the ultrathin layered protein-polyion films. Good fits were obtained for the square wave voltammograms of these films with a square wave voltammetry model featuring Eo′ dispersion, giving the average Eo′ and electrochemical rate constant (k′ s) for the proteins. ^ Human liver cytochrome P450 1A2 (hCYP1A2), cytochrome P450cam (cyt P450cam), and putidaredoxin (Pdx) were produced using genetically engineered bacteria. Improved growth and purification methods gave higher yields and lower cost compared to conventional methods. ^ Films of hCYP1A2 and polyions were used for electrochemically-driven catalytic oxidation of styrene to styrene oxide. A turnover number of 15 mol product/mol hCYP1A2/hr was found, which is much higher than those using cyt P450cam in solution and films. Results suggest that human liver cytochrome P450 1A2 is a much better catalyst for styrene oxidation than bacterial cyt P450cam.^

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