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Abstract
Chronic inflammation is associated with numerous human diseases. During inflammation, leukocytes release the enzyme myeloperoxidase (MPO) which generates reactive oxygen species such as hypochlorous acid (HOCl). Additionally, MPO generates reactive nitrogen species. These reactive species can damage host fats, proteins, and DNA, contributing to disease pathology. Because of the reactivity and short half-lives of reactive species, measurement of surrogate markers is necessary to determine their extent and source. Chlorination of the tyrosine phenol ring by HOCl to produce 3-chlorotyrosine (3ClTyr) or nitration of the phenol ring by reactive nitrogen species to produce 3-nitrotyrosine (NO2Tyr) are two such markers. Both are stable byproducts of MPO activity and are readily measurable. Some studies, however, have called into question their use as biomarkers of inflammation. Concentrations of 3ClTyr or NO2Tyr reportedly decrease upon exposure to HOCl, suggesting that any measurement of these in vivo would be underestimated. These studies, however, failed to quantify the rate of degradation and did not identify the products. Furthermore, there is some evidence to suggest that, in vivo, chlorination of tyrosine by HOCl occurs through chloramine intermediates rather than directly. This is evidenced by the preferential chlorination of tyrosine residues that are nearby lysine or histidine, whose side-chain amines react with HOCl to become chloramines. This contradicts the relatively slow reactions kinetics of tyrosine chlorination by histidine or lysine chloramine and warrants further investigation. In these studies, we identify the product of 3ClTyr and NO2Tyr reacting with HOCl as 3,5-dichlorotyrosine (Cl2Tyr) and 3-chloro-5-nitrotyrosine (ClNO2Tyr), respectively. The second-order rate constants of the chlorination of tyrosine, 3ClTyr, and NO2Tyr by HOCl, histidine chloramine, and lysine chloramine are reported. The relevance of Cl2Tyr and ClNO2Tyr in vivo are discussed. Additionally, we investigate the kinetics of tyrosine chlorination in the context of a lysine- or histidine-containing peptide. The rate of chlorination of tyrosine within a peptide is dependent on the primary and secondary structure and is a first-order, intramolecular reaction. These studies further support the role of chloramines in the chlorination of protein-bound tyrosine and, to our knowledge, are the first to provide rate constants
