Doctoral Dissertations

Title

Electrochemical and spectroscopic studies on organized films of redox proteins and surfactants

January 1997

Keywords

Chemistry, Analytical|Chemistry, Biochemistry

Ph.D.

Abstract

This thesis examines structures, electrochemical properties, and applications of new biomembrane-like films containing redox proteins and enzymes. A simple but potentially general method was developed during this thesis to prepare protein surfactant films by casting a small amount of a mixture of surfactant vesicle dispersions and proteins onto an electrode. Greatly enhanced electron transfer rates between electrodes and myoglobin (Chapter 2) or cytochrome P450$\rm\sb{cam}$ (Chapter 5) in these thin films were obtained. A formal-potential-dispersion model for these films was developed and non-linear regression analysis was used successfully to fit the model onto the square wave voltammograms of these films. Apparent electron transfer rate constant k$\rm\sb{s},$ electron transfer coefficient $\alpha,$ formal potential $\rm E\sp{o},$ and surface concentration $\Gamma$ were estimated simultaneously by the fitting procedure. These films also catalyzed the reduction of trichloroacetic acid.^ Electrochemically-driven oxidation of styrene to styrene oxide by cyt P450$\rm\sb{cam}$ via hydrogen peroxide will be discussed in Chapter 6. This system eliminates the need for the electron transfer proteins putidaredoxin and putidaredoxin reductase, and the electron donor NADH, which are required in the natural catalytic cycle of cyt P450$\rm\sb{cam}.$ Therefore, the electrochemical method greatly simplifies the catalytic system of the enzyme. The degradation of the enzyme was also investigated.^ The orientation of myoglobin in cast films of didodecyldimethylammonium bromide, dihexadecyl phosphate, and phosphatidylcholines was quantitatively investigated by linear dichroism in chapter 3. These amphiphiles were chosen to provide cationic, anionic, and zwitterionic head groups, respectively. Results show that the heme group of myoglobin in these films achieved a similar orientation irrespective of the type of surfactant in the film.^ The influence of pH on electrochemistry and protein conformation monitored by electronic and FT-IR absorption spectra will be examined for Mb-surfactant films in Chapter 4. Results suggested that electron transfer to native MbFe(III)-H$\sb2$O in thin films of surfactants was coupled to protonation at pH $>$ 5. At pH $<$ 4.6, protonation of native MbFe(III)-H$\sb2$O in the films yielded a stable, partly unfolded conformer which appears to be the main electron acceptor. ^

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