Master of Science in Chemistry, Youngstown State University, 2020, Department of Chemistry

Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules and radicals mainly produced from the partial reduction of molecular oxygen. ROS have been associated with aging and several diseases such as atherosclerosis and cancer. Metal-catalyzed oxidation (MCO) systems are systems that produce free radicals using transition metal ions such as copper or iron and hydrogen peroxide. As such, MCO may cause oxidation of proteins. To study the correlation between protein structure and oxidative damage of proteins by MCOs, different mutants of hen egg white lysozyme (HEWL) have been developed. This research focuses on the optimization of the expression of the mutant HEWL H15S. The Pichia pastoris expression system was adapted for the expression of HEWL H15S. The P. pastoris X-33-pPICZαA-hewlH15S strain was subjected to different growth conditions in a glycerol and methanol buffered media under conditions for small scale expression. Both intracellular and extracellular protein expression were analyzed for enzyme activity. Increasing glycerol concentration from 0.5% to 1% did not show significant increase in yeast growth resulting in low protein concentration and enzyme activity at 28 °C. Also, protein expression at three different methanol concentrations at 28 °C: 0.5% (v/v), 1% (v/v), and 2% (v/v) showed an increase in enzyme activity but only small changes in total protein concentration. The addition of calcium chloride showed a significant effect on the expression of H15S to about 1mg/mL compared to the other conditions without CaCl2. Lysing of the cells grown at 28 °C for intracellular analysis by the Bradford assay showed a significant band of protein corresponding to the size of the H15S mutant. A lower temperature of 22 °C at different growth and expression conditions measured high protein concentration and an increase in enzyme activity for extracellular expression. Intracellular analysis on protein expression at 22 °C measured no lysozyme acti (open full item for complete abstract)

Committee: Michael Serra PhD (Advisor); Nina Stourman PhD (Committee Member); John Jackson PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Chemistry; Microbiology

Master of Science, The Ohio State University, 1969, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, University of Akron, 2021, Polymer Science

Hydroxyapatite nanoparticles (HAp NPs) are widely used as protein and drug delivery systems since they have excellent biocompatibility. During the protein loading process, HAp NPs may change the secondary structures of the proteins, which may affect their biological functions. Previous studies have shown that using citric acid (CA) to functionalize HAp NPs is a good approach to increase the loading capacity of positively charged proteins, such as Lysozyme (LZM). However, the effects of citric acid concentration and aging time on the particle morphology, surface charge, LZM loading capacity, and LZM conformation are still not fully understood. Herein, citric acid-functionalized hydroxyapatite nanoparticles (CA HAp NPs) were synthesized, characterized, and loaded with antimicrobial protein, LZM. Morphological changes of the HAp NPs were observed from needle-like to biomimetic plate-like structures as CA concentration and the aging time were increased, along with an increase in negative surface charge. The LZM loading capacity was also greatly enhanced by increasing the citric acid content. Interestingly, no significant conformational changes were observed upon adsorption to or upon desorption from CA HAp NPs. This suggests that a higher concentration of CA leads to a greater negative surface charge of HA NPs, which increases LZM loading capacity while maintaining its biological function during the adsorption/release process. The antimicrobial test reveals that LZM has the antimicrobial ability to kill the Streptococcus mutans (S. mutans), which is the most important Gram-positive bacterium responsible for dental caries development around dental restorative implants. The LZM-loaded CA HAp NPs developed here have potential applications as antimicrobial fillers in dental composites used for restoratives as well as for broader protein delivery systems in other biomedical applications.

Committee: Nita Sahai (Advisor); Abraham Joy (Committee Member) Subjects: Biomedical Research; Materials Science

Doctor of Philosophy, University of Akron, 2018, Chemistry

Numerous nuclear magnetic resonance (NMR) experiments for observing protein-ligand interactions have been proposed over the years since NMR was first used to study biomolecules. In the work that follows, a survey of the most widely used methods is presented with applications to three protein systems. First, orthologous glutaredoxins from homo sapiens and some bacterial species were compared structurally to assay their binding interactions in a fragment-based drug discovery project. This analysis yielded ortholog specific binders to be considered as antibiotic drug leads against the glutaredoxins from Brucella melitensis and Pseudomonas aeruginosa. Next, a pedagogical research initiative was conducted along with an undergraduate biochemistry laboratory course to describe the folding and iron-sulfur cluster binding properties of the mitochondrial membrane protein mitoNEET. The students created several mitoNEET mutants and used 1D and 2D NMR experiments to characterize their effect on mitoNEET's structure and ligand binding capabilities. Finally, the lysozyme isolated from hen egg whites was observed catalyzing the polymerization of a polyacetylene. This marked the first time a hydrolase was observed catalyzing a synthetic polymer. NMR, along with X-ray crystallography, was used to describe the reaction mechanism, kinetics, and properties of the polymer product.

Committee: Christopher Ziegler Dr. (Advisor); Leah Shriver Dr. (Committee Member); Sailaja Paruchuri Dr. (Committee Member); Aliaksei Boika Dr. (Committee Member); Abraham Joy Dr. (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Biophysics; Cellular Biology; Chemistry; Medicine; Microbiology; Molecular Biology; Molecular Chemistry; Molecules; Organic Chemistry; Pedagogy; Pharmaceuticals

Master of Science, The Ohio State University, 2017, Animal Sciences

Rumen ciliates are the only predators in the rumen. They engulf ruminal bacteria and other microbes and use the microbial protein as a source of nitrogen and other nutrients after digesting the ingested bacteria using digestive enzymes contained in lysosomes. Both genes and mRNAs coding for lysozyme and peptidases including serine peptidase, metallopeptidase, and cysteine peptidase were found in Entodinium caudatum (E. caudatum), which represents the one of most predominant ruminal ciliates (Williams and Coleman, 1992). Lysozyme is a small and stable enzyme that lyses bacterial cells by breaking bacterial cell walls. Enzymatically, lysozyme hydrolyzes the 1,4-beta linkages between N-acetylmuramic acid and N-acetyl D-glucosamine residues in peptidoglycan. Serine peptidase, metallopeptidase, and cysteine peptidase are proteases that cleave specific peptide bonds of proteins. By degrading microbial proteins in the rumen, rumen ciliates drive intra-ruminal nitrogen recycling, which decreases nitrogen utilization efficiency in ruminant animals. It was hypothesized that inhibition of lysozyme and peptidases could inhibit growth of rumen E. caudatum, and thereby potentially improve nitrogen utilization by ruminants. Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases using E. caudatum as a model protozoan species. In the first in vitro experiment (chapter 3), two inhibitors each of lysozyme, serine peptidase, metallopeptidase, and cysteine peptidase at varying concentrations were tested for their inhibition to E. caudatum in vitro. The results of the first in vitro experiment (chapter 3) show that all the tested inhibitors including imidazole, phenylmethylsulfonyl fluoride (PMSF), Pefabloc®, phosphoramidon disodium salt, bestatin, and iodoacetamide reduced (p < 0. 01) counts of E. caudatum at 24 and 48 h incubation in vitro. The increased doses of each inhibitor all caused linearly (p < 0.01), quadratic (p < 0. (open full item for complete abstract)

Committee: Zhongtang Yu (Advisor); Jeffrey Firkins (Committee Member); Alejandro Relling (Committee Member) Subjects: Animal Sciences

Doctor of Philosophy, The Ohio State University, 1954, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Master of Science, Miami University, 2016, Chemistry and Biochemistry

Biomacromolecules made by covalent attachment of polymers to the surfaces of proteins are an interesting area of research with significant applications in drug therapies and growing interest in energy and nanotechnology. Protein-polymer conjugates are typically synthesized by either the grafting-to or grafting-from approach. In grafting-to, polymer is separately synthesized and characterized prior to linkage to the protein. However, in grafting-from, an initiating group is attached to the protein, which allows for growth of the polymer directly from the protein surface. Herein, a hybrid approach is used, combining the advantages of both conjugation techniques. RAFT polymerization is used to grow water soluble polymers for grafting-to and subsequent chain extension from enzymes of interest. First, conjugates made from the well-studied, model enzyme lysozyme are discussed. Next, cellulase-polymer conjugates with potential applications in biofuel production are explored. In these studies, the impact of polymer size and functional groups on enzymatic activity, thermal and chemical stability are investigated. Important trends gleaned from this work contribute to the understanding of structure-property correlations in protein-polymer conjugates. These relationships are critical to the advancement of protein-polymer conjugates in efforts to produce engineered biomacromolecules with tunable behaviors for specific applications.

Committee: Dominik Konkolewicz (Advisor); C. Scott Hartley (Committee Chair); Richard C. Page (Committee Member); Jason A. Berberich (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry; Polymer Chemistry

Doctor of Philosophy, University of Akron, 2016, Chemistry

99mTc is the most used radionuclide in diagnostic nuclear medicine. Technetium small molecule imaging agents are being used to image almost every tissue in the body, from heart and bones, to the difficult imaging of brain tissue. Though there are many 99mTc complexes currently in use, the search for an ideal technetium radiopharmaceutical is ongoing. Ideal imaging agents must be robust enough to withstand complex biological conditions and ensure clearance from the body, yet selective enough to reach their target tissues for successful scanning. Recent work has shown that compounds based on the fac-99mTc(I)(CO)3+ core exhibit increased stability and resistance to decomposition. The chemistry and pharmacokinetics of these complexes can be safely explored using the non-radioactive, isoelectronic Re(I)(CO)3+ species. Medically useful isotopes of rhenium, 188Re and 186Re, add to the potential applications of this chemistry. In this dissertation we describe the synthesis and metathesis of [Re(CO)3(TAME)]X (where X= Br-, Cl-, NO3-, ClO4-, and PF6-), and it's toxicity in both primary and immortal cell culture lines. The toxicity of the parent molecule, [Re(CO)3(OH2)3]Br, in HeLa-S3 cell culture is explored. The preparation and characterization of a Re(CO)3+-lysozyme adduct is discussed. Additional studies of the efficacy of [Re(CO)3(OH2)3]Br as an X-ray contrast agent and the preparation and partial characterization of an Re(CO)3+-insulin adduct is also described herein.

Committee: Christopher Ziegler Dr. (Advisor); Thomas Leeper Dr. (Committee Member); Sailaja Paruchuri Dr. (Committee Member); Leah Shriver Dr. (Committee Member); Todd Blackledge Dr. (Committee Member) Subjects: Biochemistry; Chemistry; Inorganic Chemistry

Master of Science in Chemistry, Youngstown State University, 2015, Department of Chemistry

Reactive oxygen species (ROS) are molecules or radicals which are produced from oxygen. Most ROS are highly reactive due to the presence of unpaired electrons in their valence shell. Metal ions such as Fe2+ or Cu+ are used as reducing agents in metal catalyzed oxidation (MCO) systems that generate the hydroxyl radical in a reaction with hydrogen peroxide. To study the oxidation of proteins by MCO systems a N103H mutant of hen egg white lysozyme (HEWL) was generated. The HEWL mutant N103H gene was successfully cloned into the pPICZαA plasmid and subsequently transformed into Mach1™-T1R chemically competent Escherichia coli. The recombinant plasmid was isolated, linearized, and transformed into the X-33 strain of the yeast Pichia pastoris by electroporation. Small scale expression was performed using a buffered glycerol media and methanol media at pH 6.0 and 7.0 for 4 days. Expression in unbuffered media was also performed. SDS-PAGE analysis of the supernatant samples from small scale expression revealed extracellular expression of a protein of the right size in a buffered medium whereas unbuffered medium showed no evidence of the expression of any proteins. Small scale expression in buffered media at pH 7.0 with antifoam appeared to give the best protein expression. A Bradford assay indicated the extracellular expression of protein to a concentration of about 0.1 mg/mL. Enzyme assays of pooled and concentrated fractions collected over four days showed no lysozyme activity. A low concentration of the N103H HEWL mutant might be the reason for no activity.

Committee: Michael Serra PhD (Advisor); Timothy Wagner PhD (Committee Member); Jonathan Caguiat PhD (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2014, Engineering and Applied Science: Chemical Engineering

Proteomics has emerged as the next phase of understanding diseases and ailments after the completion of the human genome project. There are countless examples of protein deficiencies leading to serious ailments. Therefore, proteins have been studied as biotherapeutics and have potential applications in the drug discovery process. The advancement of such biological applications demands simultaneous advancement in protein separations on both the analytical and preparative scales. Current challenges include reducing cost and increasing the speed and the yield of production while maintaining the structure of the proteins. The current standard separation method used in the industry is liquid chromatography. It is one of the few scalable, nondestructive methods that can be used for both analytical and preparative separations. The stationary phases and proteins used in this research are based on industry and research practices. The most commonly-used stationary phase material is silica, due to its chemical and mechanical stability. Chemical functionalizations of silica surfaces, which are heavily documented, allow control of the strength and nature of interactions with the stationary phase. Thus, silica was used as a base material. Similarly, lysozyme is a commonly-used sample material due to its low cost and availability. Consequently, there is a wealth of scientific literature on the properties lysozyme, and it is used as the test protein for the majority of the research reported here. Silica was functionalized with amino acids using a peptide synthesis method and the effect of amino acid functionalization on lysozyme adsorption was determined. The net charge difference between the surface and the adsorbed protein was the main driving force for the adsorption of lysozyme onto carboxyl- and amine-functionalized silica. Net charge difference did not affect lysozyme adsorption onto amino acid-functionalized silica. Rather, specific interactions between lysozyme and the (open full item for complete abstract)

Committee: Stephen Thiel Ph.D. (Committee Chair); Neville Degouvea-Pinto Ph.D. (Committee Member); Dionysios Dionysiou Ph.D. (Committee Member); Joo Youp Lee Ph.D. (Committee Member); Peter Panagiotis Smirniotis Ph.D. (Committee Member) Subjects: Chemical Engineering

Master of Science in Chemistry, Youngstown State University, 2012, Department of Chemistry

Reactive oxygen species (ROS) are compounds that are produced by the reduction of molecular oxygen; these species can generate free radicals. Metal-catalyzed oxidation systems are experimental systems that use a metal ion, such as Fe2+ or Cu+,hydrogen peroxide, and often a reducing agent to generate a free radical. The purpose of this research is to generate a suite of four site-directed mutants that were used for transformation into Pichia pastoris for small-scale expression of mutant lysozyme. The mutant lysozymes expressed will be used in future oxidation studies to determine the relationship between the site of oxidation and the level of protein structure. Two of the mutant genes, N103H and N77H, were created using the Polymerase Chain Reaction and the mutations were confirmed by gene sequencing. The genes were ligated into the pPICZα A plasmid, which supported replication in both bacterial and yeast systems. The N103H gene was successfully cloned into the yeast plasmid. Two previously prepared clones in pPICZα A, H15S and H15S+N77H, were transformed into Mach1¿¿¿¿¿¿¿-T1R chemically competent Escherichia coli. Microgram amounts of the cloned plasmids were generated, and the plasmids were linearized and then transformed into Pichia pastoris by electroporation. The phenotype of the yeast colonies was determined and small-scale expression experiments were performed. The proteins were analyzed by SDS-PAGE and by a turbidimetric assay. One overnight growth in YPD media containing Zeocin followed by a five-day growth in YPM media with an initial concentration of 2% methanol was determined to give the highest expression of a protein that retained lysozyme activity.

Committee: Michael Serra Ph.D. (Advisor); Nina Stourman Ph.D. (Committee Member); David Asch Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

PhD, University of Cincinnati, 2005, Medicine : Molecular and Cellular Physiology

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria following infection of transgenic mice. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysMD53S) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysMD53S in the respiratory epithelium of wild type (LysM+/+/LysMD53S) or lysozyme M null mice (LysM-/-/LysMD53S) resulted in elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM-/-mice that was completely reversed by targeted expression of LysMD53S. These results indicate that the muramidase activity of lysozyme is not essential for bacterial killing in vitro or in vivo.

Committee: Dr. Timothy Weaver (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2011, Chemical and Biomolecular Engineering

For bulk production of high purity protein drugs to meet FDA standards, pharmaceutical industries typically process proteins by repeated precipitations and crystallizations. The solution conditions for protein phase separation are routinely determined by trial and error. A thermodynamic model of underlying weak protein-protein interactions will help in a rapid screening of solution conditions in obtaining protein crystals of desired quality and stability. The principal objective of the thesis is, therefore, to understand the fundamental molecular level interactions among different components of protein solutions: protein, water and co-solvents, in particular, the role of hydration and co-solvent preferential interactions on protein-protein interactions. Spatial heterogeneities in protein chemistry and surface topography results in uneven specific hydration of protein surface, which alters the protein-protein interactions by eliminating some complimentary configurations. The dynamics of water at such specific hydration sites was examined in terms of average water residence times and average vacancy times and found to have little impact on protein-protein interactions. The influence of local heterogeneities in surface charge and surface roughness on specific hydration and water dynamics have also been examined. A detailed investigation of the effect of surface curvature on hydration revealed that hydration of a concave surface is thermodynamically expensive than the hydration of a chemically equivalent convex surface. The concave surface is found to remain hydrated only when the interaction between the water and constituent surface atoms are attractive. Protein phase separation is typically induced by adding a precipitating agent or co-solvent, such as inorganic salts or organic compounds: alcohols, polyols, or polyethylene glycol (PEG). Addition of a cosolvent to protein solutions alter the preferential hydration of proteins depending upon its affinity to interact (open full item for complete abstract)

Committee: Michael E. Paulaitis PhD (Advisor); Aravind Asthagiri PhD (Committee Member); Dilip Asthagiri PhD (Committee Member); Sherwin J. Singer PhD (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2010, Chemical and Biomolecular Engineering

The nanoporous PCL membranes were prepared via the combination of thermally- and nonsolvent-induced phase separations. For the phase separation process, nonsolvent has significant effect on pore formation and drug release rate. In nonsolvent-induced phase separation, a large amount of nonsolvent was added to casting solutions in order to improve pore connectivity within the membrane. The use of a Teflon plate for membrane casting can result in uniform nanoporous membranes and consistent lysozyme diffusion. Pore connectivity was improved significantly when coagulation bath temperature was lowered. By using a 5°C water coagulation bath in the wet-process precipitation, the average pore size reduced from 90 nm to 55 nm while increasing the casting solution concentration from 15 wt% to 25 wt% PCL. Thus, by varying the polymer concentration of the casting solution, the lysozyme release rate can be manipulated with precise control. The potential application of nanoporous PCL membranes to achieve the preferable zero-order release rate is demonstrated in this dissertation. Along with achieving the zero-order release rate, the nanoporous PCL membranes also provide immunoprotection for cell-based therapies/devices. Immunoisolation can be achieved by preventing Immunoglobulin G (IgG) from diffusing through the nanoporous PCL membranes. With appropriate pore size, the nanoporous PCL membranes can allow the diffusion of therapeutic agents (lysozyme) and block the diffusion of immune molecules (IgG). The application of the nanoporous PCL membranes to cell-based therapies/devices is also demonstrated in this dissertation. Extensive fibrosis induced by the healing process can be detrimental to the long-term performance of implantable applications. The prevention of fibroblast adhesion to the nanoporous PCL membrane surface is crucial for constant and well controlled drug release. This study shows a novel method to modify the nanoporous PCL membrane surface with poly(ethylene glyco (open full item for complete abstract)

Committee: W.S. Winston Ho (Advisor); Boyaka Prosper N. (Committee Member); Koelling Kurt W. (Committee Member); Lee L. James (Committee Member) Subjects: Biomedical Research; Chemical Engineering; Engineering; Polymers

Master of Science, Miami University, 2012, Zoology

The immune system is critical to an animal's survival. However, changes in immune function during insect development, and the factors that regulate these changes, are not well understood. Hemocytes carry out cellular defenses such as phagocytosis, nodulation, and encapsulation of pathogens. The enzyme phenoloxidase (PO) plays a key role in this process while the enzyme lysozyme breaks down bacterial cell walls. Total PO (TPA) and lysozyme activities, total blood hemocytes, and encapsulation ability were examined from late nymphal stages through early adulthood in the cricket, Acheta domesticus. TPA increased with age while encapsulation ability tended to decrease. The effects of crowding and light stress during nymphal development on adult immune parameters were determined. Crowding increased TPA in crickets housed in small but not large groups. Light stress negatively impacted survival, but not immune function. Further investigation of the effects of early life stress on adult immune function is thus warranted.

Committee: Kathleen A. Killian PhD (Advisor); Nancy G. Solomon PhD (Committee Member); Ann L. Rypstra PhD (Committee Member) Subjects:

Master of Science, Miami University, 2009, Chemistry and Biochemistry

This thesis describes protein structural changes in alkylammonium formate (AAF) ionic liquids. These proteins are dissolved in methylammonium formate (MAF), ethylammonium formate (EAF), and choline formate ionic liquids for study by fluorescence, circular dichroism, and UV-Visible spectroscopy. At room temperature, AAF solutions can maintain the native structure of proteins, such as cytochrome c and lysozyme, in relatively high ionic liquid concentrations (50%-70% AAF/water or AAF/phosphate buffer pH 7.0) compared to similar solutions of the organic solvents, methanol or acetonitrile, with water or buffer. As temperature increases to 80 °C, AAF denatures proteins less than organic solvents. About 1/3 of the enzyme activity of cytochrome c in 80% AAF/water can be maintained as compared to phosphate buffer. This fundamental biophysical information shows that AAFs have potential application as organic or salt solvent replacements for the separation of proteins in their native form by reversed phase or hydrophobic interaction liquid chromatography.

Committee: Neil D. Danielson (Advisor); Andre&#8217;J. Sommer (Committee Chair); Thomas L. Riechel (Committee Member); Shouzhong Zou (Committee Member) Subjects: Analytical Chemistry

Doctor of Philosophy, University of Akron, 2009, Polymer Science

The aim of this dissertation is to study the structure and dynamics of proteins immersed in trehalose – water and glycerol-water binary mixtures which are known to have bio-protective properties. Fully atomistic molecular dynamics simulations were employed for the study. Hen Egg-White Lysozyme was used as the model protein.In the first part of the study the behavior of the protein in 0, 10, 20, 30 and 100% by weight trehalose-water binary mixtures was studied. The global structure of the protein in the mixtures was found to be insensitive to the concentration of trehalose while the local structure showed a strong dependence on it. The dynamic behavior of the protein, as quantified by the Incoherent Intermediate Scattering Function, showed a non-monotonic dependence on trehalose content. Indeed, in the case of 30% trehalose – 70% water mixture the protein relaxes faster than in the case of 20% trehalose – 80% water mixture. This counterintuitive behavior is rationalized in terms of the behavior of the solvent - protein hydrogen bonds and indicates the onset of preferential hydration. The dynamic behavior of the hydration layer was also explored. The short-length-scale dynamics of this layer were found to be insenstive to trehalose concentration. However, the long-length-scale behavior showed a significant dependence on trehalose content. Insights into the dynamics of bound and mobile hydration water are also presented. In the second part of the study the behavior of the protein in 0, 10, 20, 30 and 100% by weight glycerol-water binary mixtures was studied. Similar to the case of trehalose-water binary mixtres, the global structure of the protein was found to be insensitive to the concentration of glycerol while the local structure showed a strong dependence on it. The dynamic behavior of the protein showed a monotonic dependence on glycerol content. The fluctuations of the protein residues with respect to each other were found to be similar in all water-containing (open full item for complete abstract)

Committee: Gustavo Carri Dr. (Advisor) Subjects: Biophysics; Polymers

Master of Science in Chemistry, Youngstown State University, 2013, Department of Chemistry

Protein oxidation has been correlated with several chronic diseases including Alzheimer's disease, Parkinson's disease, and cataractogenesis. The purpose of this project was to isolate and characterize the various oxidized forms of hen egg white lysozyme that were produced by a copper(II)/hydrogen peroxide metal-catalyzed oxidation system. Five oxidized protein variants were purified using high performance liquid chromatography on a cation-exchange column. Tandem mass spectrometry determined that several amino acids were oxidized in each variant with histidine 15 being the most readily oxidized residue. Bacteriolytic assays showed decreased activity of Peaks IB, IIB, and III (31.4%, 61.2%, and 86.5%, respectively) relative to native enzyme while the activity for Peaks IV and V was greater than that of native enzyme (215% and 308%, respectively). Crystals of Peaks IB, III, IV, and V were grown, but attempts to determine the crystal structure were unsuccessful.

Committee: Michael Serra PhD (Advisor); Nina Stourman PhD (Committee Member); Gary Walker PhD (Committee Member) Subjects: Biochemistry