Abstract
Oxidative reactions that modify proteins have been implicated in the pathogenesis of aging and disease (1). It has been difficult to identify the physiologically relevant pathways, however, because the reactive intermediates are short-lived. We attempt to determine which oxidative pathways damage proteins in vivo by first identifying stable end products of potential pathways through in vitro experiments. We then analyze normal and diseased tissues for those compounds. For example, two stable isomers of p-tyrosine—ortho-tyrosine and meta-tyrosine—appear after hydroxyl radical modifies protein-bound phenylalanine residues (2–4). In contrast, o,o′-dityrosine forms when hydroxyl radical crosslinks tyrosine residues. o,o′-Dityrosine also appears when free or protein-bound tyrosine is attacked by tyrosyl radical (5), which is produced from tyrosine and H2O2 by the heme enzyme myeloperoxidase (6,7). Tyrosyl radical does not generate ortho-tyrosine and meta-tyrosine, however (2–5). Another oxidant, hypochlorous acid (HOCl), produces 3-chlorotyrosine when it reacts with tyrosine (8,9). HOCl is generated only by myeloperoxidase, which requires H2O2 and Cl− to perform the reaction. Thus, determining relative levels of ortho-tyrosine, meta-tyrosine, o,o′-dityrosine, and 3-chlorotyrosine can indicate which pathway might have inflicted protein damage in vivo in a particular tissue. These amino acid products are useful markers because they are stable to acid hydrolysis, an essential analytical step.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shigenaga, M. K., Hagen, T. M., and Ames B. N. (1994) Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91, 10,771–10,778.
Pennathur, S., Wagner, J. D., Leeuwenburgh, C., Litwak, K. N., and Heinecke, J. W. (2001) A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J. Clin. Invest. 107, 853–860.
Leeuwenburgh, C., Rasmussen, J. E., Hsu, F. F., Mueller, D. M., Pennathur, S., and Heinecke, J. W. (1997) Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J. Biol. Chem. 272, 3520–3526.
Pennathur, S., Jackson-Lewis, V., Przedborski, S., and Heinecke, J. W. (1999) Mass spectrometric quantification of 3-nitrotyrosine, orthortho-tyrosine, and o,o′-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson’s disease. J. Biol. Chem. 274, 34,621–34,628.
Heinecke, J. W., Li, W., Francis, G. A., and Goldstein, J. A. (1993) Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins. J. Clin. Invest. 91, 2866–2872.
Heinecke, J. W., Li, W., Daehnke, H. L. 3rd, and Goldstein, J. A. (1993) Dityrosine, a specific marker of oxidation, is synthesized by the myelo-peroxidase-hydrogen peroxide system of human neutrophils and macrophages. J. Biol. Chem. 268, 4069–4077.
Jacob, J. S., Cistola, D. P., Hsu, F. F., Muzaffar, S., Mueller, D. M., Hazen, S. L., and Heinecke, J. W. (1996) Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway. J. Biol. Chem. 271, 19,950–19,956.
Hazen, S. L., Hsu, F. F., Mueller, D. M., Crowley, J. R., and Heinecke, J. W. (1996) Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J. Clin. Invest. 98, 1283–1289.
Hazen, S. L. and Heinecke, J. W. (1997) 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is arkedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075–2081.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.,Totowa, NJ
About this protocol
Cite this protocol
Heinecke, J.W. (2003). Isotope Dilution Gas Chromatography-Mass Spectrometric Analysis of Tyrosine Oxidation Products in Proteins and Tissues. In: Hensley, K., Floyd, R.A. (eds) Methods in Biological Oxidative Stress. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-424-7:93
Download citation
DOI: https://doi.org/10.1385/1-59259-424-7:93
Publisher Name: Humana Press
Print ISBN: 978-0-89603-815-8
Online ISBN: 978-1-59259-424-5
eBook Packages: Springer Protocols