Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.
Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.
Linetsky
M
M
Mason Eye Institute-East, University of Missouri, Columbia 65201, USA. opthml@showme.missouri.edu
LeGrand
R D
RD
Mossine
V V
VV
Ortwerth
B J
BJ
D002241:Carbohydrates / Q000737:chemistry; D003432:Cross-Linking Reagents; D003543:Cysteamine / Q000031:analogs & derivatives / Q000737:chemistry; D017127:Glycation End Products, Advanced / Q000138:chemical synthesis* / Q000737:chemistry; D006031:Glycosylation; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry*; D015416:Maillard Reaction; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D002241:Carbohydrates; D003432:Cross-Linking Reagents; D017127:Glycation End Products, Advanced; D003543:Cysteamine; D012685:Sepharose; C013411:biocytin; D008239:Lysine