Site-specific glycation of lens crystallins by ascorbic acid.
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 NaCNBH3. 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 crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated 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 epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Slight
S H
SH
Prabhakaram
M
M
Sun
Y
Y
Smith
J B
JB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D001894:Borohydrides; D003459:Crystallins; D046911:Macromolecular Substances; D010446:Peptide Fragments; C009282:sodium cyanoborohydride; D002918:Chymotrypsin; D010434:Pepsin A; D005947:Glucose; D001205:Ascorbic Acid