Sarah - [Frontiers in Bioscience 6, a17-24, August 1, 2001]...

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[Frontiers in Bioscience 6, a17-24, August 1, 2001] 17 REACTIVE CARBONYL FORMATION BY OXIDATIVE AND NON-OXIDATIVE PATHWAYS Sarah Adams, Pattie Green, Renee Claxton, Sabrina Simcox, Michelle V. Williams, Katherine Walsh and Christiaan Leeuwenburgh Biochemistry of Aging Laboratory, University of Florida, Box 118206, Gainesville, FL 32611 TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Materials and Methods 3.1. Materials, reagents, and isolation of proteins 3.2. Metal-catalyzed protein oxidation 3.3. Protein modification by aldehydes 3.4. Protein oxidation by hypochlorous acid and peroxynitrite 3.5. Determination of reactive carbonyls in proteins 4. Results 4.1. Metal-catalyzed oxidation 4.2. Carbonyl formation by non-oxidative mechanisms 4.3. Carbonyl formation by the strong oxidants hypochlorous acid and peroxynitrite 4.4. Hemoglobin, myoglobin, and cytochrome c have a similar absorbance spectrum to DNPH 5. Discussion 6. Acknowledgements 7. References 1. ABSTRACT The spectrophotometric protein carbonyl assay is used as an indicator of protein damage by free radical reactions in vitro and in a variety of pathologies. We investigated model proteins and a variety of oxidative and non-oxidative reactions, as well as what effects hemoglobin, myoglobin, and cytochrome c might have on levels of protein carbonyls. We show that oxidative as well as non-oxidative mechanisms introduce carbonyl groups into proteins, providing a moiety for quantification with 2,4-dinitrophenylhydrazine (DNPH). Bovine serum albumin exposed to oxidative scenarios, such as hypochlorous acid, peroxynitrite, and metal-catalyzed oxidation exhibited variable, but increased levels of carbonyls. Other non-oxidative modification systems, in which proteins are incubated with various aldehydes, such as malondialdehyde, acrolein, glycolaldehyde, and glyoxal also generated significant amounts of carbonyls. Furthermore, purified myoglobin, hemoglobin, and cytochrome c show high absorbance at the same wavelengths as DNPH. The high levels observed are due to the innate absorbance of hemoglobin, myoglobin, and cytochrome c near the assay spectra of DNPH. These studies show that carbonyl content could be due to oxidative as well as non-oxidative mechanisms and that heme-containing compounds may effect carbonyl quantification. 2. INTRODUCTION Oxygen free radicals are produced as byproducts of many endogenous and exogenous sources including UV light, radiation, neutrophil activity, and metabolism (1-4). Oxidative stress often leads to lipid, nucleic acid, carbohydrate, and protein modifications. Protein oxidation and modification have been shown to increase during aging and exacerbate many pathological processes, such as atherosclerosis and human cataracts (1, 3-5). Oxidation of proteins can lead to the formation of oxidized amino acids, such as dityrosine, 3-nitrotyrosine, 3-chlorotyrosine, oxohistidine, and altered amino acid side chains containing reactive carbonyls (2, 6-12). Oxidation of proteins can result in the loss of catalytic function, increased sensitivity
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This note was uploaded on 05/19/2011 for the course BCH 3218 taught by Professor Johnsteward during the Fall '08 term at University of Florida.

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Sarah - [Frontiers in Bioscience 6, a17-24, August 1, 2001]...

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