Degree Type

Dissertation

Date of Award

2002

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

James Thomas

Abstract

Cysteine residues have a reduced thiol group that frequently exerts an influence on the function of proteins. Oxidation of the cysteine through S-thiolation, S-nitrosylation, or irreversible oxidation perturbs these functions. Sulfinic and sulfonic acids produced by irreversible oxidation are exceptionally damaging since these modifications are permanent in cells. In order to detect and quantitate both protein sulfinic and sulfonic acids, it was necessary to develop an assay using a novel amino acid analysis procedure. Purified creatine kinase and carbonic anhydrase III were used to establish the method, and the assay produced results that agreed with total cysteine irreversible oxidation determined by isoelectric focusing.;Glutathione has been hypothesized to protect cysteine residues from irreversible oxidation through protein S-glutathiolation. This concept was investigated both in vitro and in vivo using carbonic anhydrase III as a model protein. The two sulfhydryls of carbonic anhydrase III were irreversibly oxidized to sulfinic acid and sulfonic acids by hydrogen peroxide and peroxyl radicals, but when glutathione was approximately equimolar to protein thiols, these modifications were prevented through S-glutathiolation. Hypochlorous acid oxidation did not produce protein S-glutathiolation and generated irreversible cysteine oxidation even in the presence of glutathione. Protection of carbonic anhydrase III thiols by S-glutathiolation was highly dependent on glutathione concentration both in vitro and in cultured rat hepatocytes. When hepatocyte glutathione was depleted with diethyl maleate, carbonic anhydrase III was more prone to irreversible oxidation following menadione stimulated oxidation. Age-related depletion of glutathione might increase irreversible cysteine oxidation, and both total protein extracts and carbonic anhydrase III were significantly more irreversibly oxidized in the liver tissues of older rats.;Experiments using creatine kinase as a model protein also demonstrated that irreversible cysteine oxidation produced by hydrogen peroxide or peroxyl radicals could be prevented by glutathione through protein S-glutathiolation. Rat skeletal muscle and heart tissue showed an approximate two-fold age-related increase in total protein sulfinic acids, with a concurrent 30--40% decrease in soluble glutathione. Irreversible oxidation of creatine kinase in these tissues followed the same trends as total protein sulfinic acids. Thus, depletion of cellular glutathione is expected to accelerate cysteine irreversible oxidation.

DOI

https://doi.org/10.31274/rtd-180813-8770

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Michael James Hamann

Language

en

Proquest ID

AAI3073450

File Format

application/pdf

File Size

135 pages

Included in

Biochemistry Commons

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