Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of endogenous (e.g., hormones, neurotransmitters, bile acids) as well as xenobiotic (e.g, drugs, environmental pollutants) compounds. Alteration in the catalytic activity of hSULT2A1 can lead to outcomes like endocrine disruptions or aberrant drug metabolism and xenobiotic toxicity. Oxidative and electrophilic stresses are known to cause physiological damage and be implicated as possible underlying pathologic mechanisms of a wide range of diseases. To examine the oxidative as well as electrophilic regulation of hSULT2A1, model oxidants (glutathione disulfide (GSSG), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), diamide, tert-butyl hydroperoxide (TBHP)) and electrophiles such as quinone metabolites of polychlorinated biphenyls (PCB-quinones) and phenyl-p- benzoquinone were chosen for this study. Mechanistic studies correlating the enzyme structural modifications with alteration in the catalytic properties were performed to elucidate the catalytic regulative mechanism of an individual oxidant or electrophile. Thiol oxidants including GSSG, DTNB, and diamide showed catalytic regulation of hSULT2A1. Changes in protein intrinsic fluorescence indicated conformational alterations in hSULT2A1 following the reaction with diamide. Binding properties of hSULT2A1 for its substrates were also altered after reaction with these thiol oxidants, which could be one major reason for the kinetic alteration due to oxidative modification. Formation of mixed disulfides with cysteines in hSULT2A1 was also identified as a result of reaction with GSSG and DTNB. TBHP was chosen as a model for lipid peroxides, and reaction with this hydroperoxide decreased the catalytic function of hSULT2A1. The dissociation constant for binding of dehydroepiandrosterone (DHEA) was significantly altered with TBHP-pretreatment, but this did not affect the binding of 3',5'-adenosine diphosphate (PAP) to the enzyme. Structural analysis identified cysteine sulfonic acids and methionine sulfoxide formation after reaction of hSULT2A1 with TBHP, which could account for the alterations in the binding properties and the catalytic activity. Both PCB-quinones and PBQ could regulate the catalytic activity of hSULT2A1. Although PCB-quinones only caused decreases in the catalytic activity at all concentrations tested, pretreatment with PBQ indicated that lower concentrations resulted in an increase in the catalytic activity of hSULT2A1 that was followed by a decrease in the catalytic activity of hSULT2A1 upon increasing the concentration of PBQ in the pretreatment. Differences in the dissociation constants of PAP after PBQ-pretreatment were also observed, indicating the key role played by these PCB-quinones in altering the binding of either PAP or the sulfuryl donors, PAPS. Adducts at cysteines in hSULT2A1 were formed following reactions with PCB-quinones and PBQ. Small amounts of cysteine sulfonic acids and methionine sulfoxides were also formed following reaction of the protein with PCB-quinones and PBQ. Therefore, alterations in both the catalytic function as well as the structural properties of hSULT2A1 by interaction with oxidants and electrophiles may lead to changes in the metabolism of xenobiotics, as well as alterations in the endogenous balance of various steroid hormones. Such changes may be an important component in physiological damage that occurs under oxidative and electrophilic stress.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.

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The effects of oxidation by sodium hypochlorite (0, 0.8, 2, and 5%, NaOCl), the presence of endogenous proteins, and amylose content on waxy and common rice flours (WF, CF) and starches (WS, CS) were investigated in terms of in vitro starch digestibility, morphology and surface properties, and thermal and rheological characteristics. The concentration of NaOCl had an effect on all the samples including WF, CF, WS, and CS. The carbonyl and carboxyl group contents increased up to 25 and 10 folds (P < 0.05) of oxidized starches (WS, CS), respectively. Only mild oxidation (P < 0.05) occurred in flours (WF, WS). In addition, endogenous proteins were oxidized according to amino acid analysis and SDS–PAGE results. Glu+Gln, Gly, His, Arg, Tyr, and Lys were more sensitive to NaOCl oxidation. Disulfide bonds, hydrophobic force, and hydrogen bonds were involved in protein polymerization after NaOCl oxidative modification. In granular state, the in vitro starch digestibility of WF, WS, and CS decreased by 5% NaOCl oxidation. After gelatinization, only 2 and 5% oxidized WS had lower digestibility. Scanning electron microscopy and confocal laser scanning microscopy further demonstrated that protein existed on the surface of starch granules and had aggregation by oxidation. X-ray diffraction patterns showed the crystallinity of 5% oxidized flours and starches was reduced compared with all their non-oxidized samples. Thermal and rheological properties were analyzed by differential scanning calorimetry and rheometer, respectively. Starch gelatinization peak temperature of flours (WF, RF) was increased by 3 °C, but starches (WS, CS) had a significantly decrease by 8 °C. Viscoelastic patterns were dramatically changed by oxidation. Oxidized WF and CF had increased in both viscosity and elasticity by oxidation, whereas both WS and CS had significantly lower viscoelasticity after oxidative modification.

Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.