Site-specific glycation of lens crystallins by ascorbic acid

• Published in: Biochimica et biophysica acta Vol. 1117; no. 2; pp. 207 - 215
• Main Authors: Ortwerth, Beryl J., Slight, Simon H., Prabhakaram, Malladi, Sun, Yiping, Smith, Jean B.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.09.1992; Elsevier
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Animals; Ascorbic acid; Ascorbic Acid - metabolism; Binding Sites; Biological and medical sciences; Borohydrides - pharmacology; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin - metabolism; Crystallins - isolation & purification; Crystallins - metabolism; Electrophoresis, Polyacrylamide Gel; Glucose - metabolism; Glycation; Glycosylation; Kinetics; Lens carboxymethylation; Lens diseases; Lens peptide; Macromolecular Substances; Mass Spectrometry; Medical sciences; Molecular Sequence Data; Ophthalmology; Pepsin A - metabolism; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; α-Crystallin; SDS-PAGE; Lens peptide; Ascorbic acid; CML; DTPA diethylenetriaminepentaacetic acid; MS; α-Crystallin; Lens carboxymethylation; Mass spectrometry; Glycation; HPLC; ASA; Animal model; Cataract; Bovine; Pathophysiology; Proteins; Eye disease; Vertebrata; Chemical modification; Mammalia; Crystallin; Lens; Artiodactyla; Ungulata; Molecular adduct
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(92)90081-5

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1- 14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH 3. This rate of incorporation was 6–7-fold more rapid than that obtained with [[ 14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the cyrstallin subunits. [1- 14]Ascorbic acid-label led α-crystallin was separated into into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N ϵ -(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.