This dissertation examines the role of reduced glutathione (GSH) in the oxidation of proteins. Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.
","abstract_html":"<p>This dissertation examines the role of reduced glutathione (GSH) in the oxidation of proteins. Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.</p>","abstract_has_math":false,"creators":["Mallis, Robert"],"institution":null,"degree_name":"Doctor of Philosophy","degree_level":"dissertation","degree_discipline":null,"degree_department":"Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology (LAS)","school":null,"contributors":[],"advisors":["James A. Thomas"],"committee_chairs":[],"committee_members":[],"year":2000,"date_issued":"2000-01-01","date_published":"2000-01-01","updated_at":"2026-07-24T02:38:23Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.31274/rtd-180813-15270"],"render_values":[{"text":"https://doi.org/10.31274/rtd-180813-15270","href":"https://doi.org/10.31274/rtd-180813-15270","code":true}]},{"key":"dc:identifier","label":"Identifier","values":["archive/lib.dr.iastate.edu/rtd/13916/"],"render_values":[{"text":"archive/lib.dr.iastate.edu/rtd/13916/","href":null,"code":true}]}]},"links":{"outbound_url":"https://dr.lib.iastate.edu/handle/20.500.12876/67442","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["James A. 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Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.
"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["A role for glutathione in reactions between oxidants and proteins"]}]}],"canonical_facts":{"dc:contributor.advisor":["James A. Thomas"],"dc:contributor.department":["Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology (LAS)"],"dc:creator":["Mallis, Robert"],"dc:date":["2018-08-23T03:01:59.000"],"dc:date.accessioned":["2020-06-30T07:32:32Z"],"dc:date.available":["2020-06-30T07:32:32Z"],"dc:date.issued":["2000-01-01"],"dc:description.abstract":["This dissertation examines the role of reduced glutathione (GSH) in the oxidation of proteins. Carbonic anhydrase III is a cytosolic protein which is S-glutathiolated in cells under oxidative stress. The pure protein was found here to be S-glutathiolated by two oxidants, 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and hydrogen peroxide (H2O2). At physiologically relevant molar ratios of GSH to protein, GSH was found to mediate protection of carbonic anhydrase III from irreversible oxidation via S-glutathiolation. Both oxidants were found to react directly with the protein. S-glutathiolation. appears to result from the formation of an activated protein thiol that reacts with GSH;H-Ras, a low molecular weight G-protein that regulates proliferation and differentiation in cells, may be regulated by oxidative events. For this reason, oxidative modifications of the cysteine residues of H-Ras were studied. The pure protein was modified on multiple thiols when incubated with thiol oxidants. H2O2 + GSH was found to S-glutathiolate H-Ras on at least one cysteine, while either diamide + GSH or glutathione disulfide (GSSG) was found to S-glutathiolate at least two H-Ras cysteines. The NO donor S-nitrosoglutathione caused S-nitrosylation of H-Ras on four cysteine residues;Within NIH/3T3 cells overexpressing H-Ras, H-Ras was S-glutathiolated on multiple thiols by diamide. At least one of the cysteine residues modified in cells by diamide is normally lipidated, suggesting a role for oxidation in regulating the membrane association of H-Ras. In NIH/3T3 cells overexpressing H-Ras, S-nitrosocysteine was found to cause both S-nitrosylation and S-glutathiolation of H-Ras. Thus, oxidative modification of H-Ras can occur simultaneously on multiple thiols and by multiple mechanisms;The effect of S-nitrosocysteine on low molecular weight thiols and soluble proteins in NIH/3T3 cells was also studied. S-nitrosocysteine was found to be an effective S-nitrosating agent, causing S-nitrosylation of glutathione and proteins. S-nitrosocysteine was also found to be an effective oxidant, causing formation of glutathione disulfide, cysteine-glutathione disulfide, cystine, S-glutathiolated and S-cysteylated proteins. The biological effects of S-nitrosocysteine are likely to be mediated not only through nitrosative, but also oxidative events. Thus cellular GSH pools interact significantly with S-nitrosocysteine and may be a factor in cell-specific variations in responses to S-nitrosothiols.
"],"dc:format.mimetype":["application/pdf"],"dc:identifier":["archive/lib.dr.iastate.edu/rtd/13916/"],"dc:identifier.doi":["https://doi.org/10.31274/rtd-180813-15270"],"dc:identifier.uri":["https://dr.lib.iastate.edu/handle/20.500.12876/67442"],"dc:language.iso":["en"],"dc:title":["A role for glutathione in reactions between oxidants and proteins"],"dc:type":["dissertation"],"thesis:degree_level":["dissertation"],"thesis:degree_name":["Doctor of Philosophy"]},"updated_at":"2026-07-24T02:38:23Z"}