Submitted on November 8, 2004
Revised on March 22, 2005
Accepted on March 31, 2005
Chemical modification of proteins during peroxidation of phospholipids: Identification of adducts and crosslinks, and evidence for increased chemical modification of plasma proteins by phospholipids in diabetes
Andrzej S. Januszewski, Nathan L. Alderson, Alicia J. Jenkins, Suzanne R. Thorpe, and John W. Baynes
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208
Corresponding Author: john.baynes{at}sc.edu
Chemical modification of proteins by advanced glycation and lipoxidation end-products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of lipoxidative modification of protein, we studied oxidation of phospholipids in the presence of the model protein RNase A, and compared protein-bound products formed in these reactions to those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to loss of amino groups in RNase and incorporation of phosphate, hexanoate, pentanedioate, nonanedioate and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate respectively, accounted for ~20% of the crosslinking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared to control subjects.
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