Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose.
We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N(epsilon)-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N(epsilon)-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.
Hinton
D J S
DJ
Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Whiteknights, Reading, UK. d.j.s.hinton@reading.ac.uk
Ames
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005632:Fructose / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry; D017127:Glycation End Products, Advanced / Q000138:chemical synthesis*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000138:chemical synthesis / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D017127:Glycation End Products, Advanced; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine; D005632:Fructose; C048496:N(6)-carboxymethyllysine; D005947:Glucose; D008239:Lysine