Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate.
N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.
Dunn
J A
JA
Department of Chemistry, University of South Carolina, Columbia 29208.
Ahmed
M U
MU
Murtiashaw
M H
MH
Richardson
J M
JM
Walla
M D
MD
Thorpe
S R
SR
Baynes
J W
JW
D001205:Ascorbic Acid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry*; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; C048496:N(6)-carboxymethyllysine; D008239:Lysine; D001205:Ascorbic Acid