Graduation date: 1997

Polyphenol oxidase (PPO) from d'Anjou pears was purified and some molecular properties were studied. The extraction of PPO was accomplished in the presence of the phenolic binder AG 2X-8 and Triton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Three fractions of pear PPO were purified by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, followed by chromatography on DEAE-cellulose and hydroxylapatite (HA). The best resolution was achieved when the columns were developed at room temperature. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to the procedure's success. The three fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide gel electrophoresis and corresponded to relative mobilities of 0.41 (HA 1), 0.43 (HA 2), and 0.73 (DEAE 3). However, other minor protein bands were noticed on lithium dodecyl sulfate, LDS, electrophoresis. Dimethylsulfoxide, DMSO, was found to significantly increase the PPO activity over the control. An 80% increase in enzyme activity was observed when 5% DMSO was added to the enzyme fraction. Chlorogenic acid reacted with one of the purified fractions (HA 2) at pH 7 to yield 5 additional PPO bands upon electrophoresis. However, no electrophoretic changes were observed with another fraction (DEAE 3) or when 2,3-dihydroxybenzaldehyde was reacted with the two PPO fractions. The molecular weights of the three multiple forms of pear PPO were similar and were 45,300 ± 5% daltons by Sephacryl S-300 gel filtration and 59,600 ± 3% daltons by LDS electrophoresis in 10% and gradient polyacrylamide slab gels. The isoelectric points of the three PPO fractions were: HA 1, pi 4.3 and 4.5; HA 2, pi 4.5 and 4.7; and DEAE 3, pI 6.9. The three fractions differed in amino acid composition with HA 1 having a higher proportion of hydrophobic amino acids and less charged residues than either HA 2 or DEAE 3. All three forms of PPO contained a high proportion of the acidic amino acids and/or their amides, and glycine. / Graduation date: 1983

Graduation date: 1981

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 110-114). Also available via the Internet.

Thesis (doctoral)--Friedrich-Wilhelms-Universität zu Berlin, 1906. / Lebenslauf. Includes bibliographical references.

No description available.

This research has been directed at the study of both the enzyme sulfite oxidase and molybdenum model chemistry. A modification of a previously published procedure has been used to purify sulfite oxidase in high yield which is well-suited for experiments requiring prosthetically intact enzyme and which is not contaminated with extraneous heme or with other redox active proteins. Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin, lumiflavin, and riboflavin semiquinone radicals with the redox centers of purified sulfite oxidase. Two distinctly different intramolecular electron transfer processes were observed between the molybdenum and heme sites of the enzyme, and these assignments were supported by flash photolysis studies of the cyanide-inactivated enzyme and the sulfite oxidase heme peptide. Microcoulometric experiments on sulfite oxidase have shown that the enzyme requires the addition of three electrons for complete subunit reduction. Midpoint potentials for the Mo(VI)/Mo(V), Mo(V)/Mo(IV), and Fe(III)/Fe(II) couples have been obtained under varied buffer conditions. The midpoint potentials obtained under High-pH and Low-pH conditions provided a means for reductively titrating the enzyme to the Mo(V) oxidation state for EXAFS studies. EXAFS of sulfite oxidase under High-pH and Low-pH conditions have provided the first example of a structural study of the three accessible oxidation states (Mo(VI), Mo(V), and Mo(IV)). A biologically relevant synthetic model for the formation of the Mo(V) Low-pH form of sulfite oxidase has been developed. The Mo(V) model compound closely resembles the minimum coordination environment for the Mo(V) Low-pH form of sulfite oxidase as determined by EXAFS. Using synchrotron radiation, molybdenum L-edge x-ray absorption spectra have been obtained for a variety of oxomolybdenum(V) compounds which serve as models for sulfite oxidase. An attempt has been made to correlate unique features of the molecules to the observed 2P → 4d electronic transitions.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from Dissertation Abstracts.

Not available / text

Graduation date: 1992