Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed on Nov. 10, 2006). PDF text: 116 p. : ill. (some col.) ; 10.31Mb. UMI publication number: AAT 3215816. Includes bibliographical references. Also available in microfilm and microfiche format.

Cytochrome c was immobilized to several supports to study the effects of immobilization on the molecule and to serve as a model for the in vivo system. Immobilization was accomplished by covalent attachment of cytochrome c to the support surface, either Sepharose 6MB or glassy carbon. The effect of the coupling conditions on the covalent attachment reaction was studied with Sepharose 6MB. The reactive groups were monitored colorimetrically and were highly susceptible to hydrolysis. Correction for hydrolysis indicated that the covalent attachment reaction was first order with respect to reacted groups. Coupling conditions most affecting the amount of attached cytochrome c were the initial cytochrome c concentration, temperature, and pH. A detailed study of the resulting immobilized cytochrome c was conducted based on its three characteristic properties: spectra, oxidation reduction potential, and biological activity. The spectral properties demonstrated that no major conformational changes had occurred upon immobilization since the spectra were essentially the same. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were found to be 20-25 mV lower than native cytochrome c (+ 270 mV). Two samples, the heaviest loaded, were approximately equal to the native protein suggesting that they may be least affected by immobilization. The biological activity measurements provide an indication of the ability of the molecule to function properly. The Michaelis constant (K(m)) for cytochrome oxidase and reductase with immobilized cytochromes c were significantly higher (20-400X) than the K(m) for soluble cytochrome c. The higher K(m)s reflect that about 1% of the immobilized cytochrome c is availble for reaction in agreement with distribution and exclusion studies. Correction of the immobilized cytochrome c K(m)s for available protein results in values similar to the soluble cytochrome c K(m). Immobilization of cytochrome c to glassy carbon was performed by two procedures employing a carbodiimide or 4-vinylpyridine as the coupling reagents. The former resulted in electrodes with higher specific activities and lower protein loadings than the latter. In both cases up to 60% of the immobilized protein was held by adsorption on the surface. Protein coverages were approximately 10⁻⁸ to 10⁻⁹ moles/cm² which corresponds to 100-800 layers.

The cytochrome P450 enzymes (CYPs) are very attractive biocatalysts because of their ability to regio- and stereo-selectively catalyze the insertion of a single atom of molecular oxygen into inactivated C-H bonds. There are many drawbacks, however, limiting the use of these enzymes in organic synthesis, including the need for expensive cofactors, low stability, and low tolerance to organic solvents. The goal of this thesis was to overcome some of these drawbacks for human CYP2D6. This isoform was selected because of its broad substrate promiscuity and high importance in drug metabolism. / We have tested inexpensive chemicals to replace the natural cofactors of CYP2D6, NADPH and cytochrome P450 reductase (CPR). The results showed that cumene hydroperoxide and tert-butyl hydroperoxide can successfully substitute CPR and NAD(P)H with retained regio- and stereo-selectivity. Moreover, with these surrogates, product formation and initial rates are increased by as much as two fold compared to the use of the natural cofactors. / It is widely accepted that even small proportions of organic solvents in the buffer can deactivate most enzymes including P450s. Our studies on the biocatalysis of CYP2D6 in organic solvent/buffer emulsions showed that under the optimized conditions, as much as 76% of the enzyme activity was retained. Product formation in biphasic solvent systems is comparable whether the natural redox partner and cofactor are used, or a surrogate. In addition, a correlation was observed between the log P and the suitability of a solvent for enzymatic activity, with higher log P resulting in higher enzymatic activity. These results were obtained with dextromethorphan (DXM), a water soluble substrate. A very hydrophobic substrate, 7-benzyloxy-4-N,N-diethylaminomethyl-coumarin (BDAC), was also tested successfully to demonstrate the utility of this method. / Lyophilization is usually required to remove water before using enzymes in nearly anhydrous solvents. This physical process is harmful to P450 enzyme activity. We therefore tested numerous sugars as lyoprotectant during lyophilization. Addition of trehalose or sucrose before lyophilization allowed the retention of 80% of the CYP2D6 activity, compared to 40% remaining activity in its absence. CYP2D6 co-lyophilized with trehalose was next tested in selected hydrophobic organic solvents in the absence of water. The enzymatic activity was found to strongly depend on the hydrophobicity of the solvent. Interestingly, the enzyme showed higher catalytic ability in n-decane or n-dodecane than in the standard buffer. This was unexpected considering that the activity of most enzymes was reported to decrease to 10% or less in nearly anhydrous organic solvents. / The last objective of this thesis was to improve the stability and/or activity of CYP2D6. Use of DNA self-assemblies to encapsulate P450 enzymes was envisaged to potentially increase their stability. Indeed, DNA assemblies have many advantages compared to traditional solid supports reported for enzymes. Our preliminary results showed that CYP2D6 templated the formation of cyclic DNA dimeric and tetrameric over polymeric self-assemblies. Characterization of the CYP2D6 activity in the presence of the DNA self-assemblies revealed no loss of activity or stability.

Thesis (M.Sc.) --University of Adelaide, 1946. / Typewritten copy.

Thesis (B.S.) Magna Cum Laude--Butler University, 2008. / Includes bibliographical references (76-77).

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