The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.
The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.
Slight
S H
SH
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Prabhakaram
M
M
Shin
D B
DB
Feather
M S
MS
Ortwerth
B J
BJ
D015083:2,3-Diketogulonic Acid / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D011107:Polylysine / Q000378:metabolism*
D000596:Amino Acids; D001894:Borohydrides; D003459:Crystallins; D011107:Polylysine; D015083:2,3-Diketogulonic Acid; C048496:N(6)-carboxymethyllysine; C009282:sodium cyanoborohydride; D008239:Lysine; D001205:Ascorbic Acid