Argpyrimidine, a blue fluorophore in human lens proteins: High levels in brunescent cataractous lenses

Purpose. To determine whether the human lens contains argpyrimidine, a modi fication of arginine by methylglyoxal, to establish how argpyrimidine conte nt relates to lens aging and cataract formation. METHODS. A monoclonal antibody was used to measure argpyrimidine by a compe titive ELISA in water soluble (WS) and insoluble (WI) lens fractions from y oung, aged, nuclear cataractous, and brunescent cataractous lenses. Brunesc ent cataractous lens proteins were digested by enzymes, the digest was subj ected to HPLC, and the eluate was analyzed for argpyrimidine. Lens proteins from aged lenses (from donors 65 to 80 years of age) were fractionated on a Sephadex G-200 column, and the crystallins were tested for argpyrimidine. RESULTS. The competitive ELISA showed two to three times as much argpyrimid ine in water-insoluble proteins as in watersoluble proteins. Although no cl ear cut increase with the age of the lens donors in either the water-solubl e or the insoluble protein fractions was found, the argpyrimidine levels in brunescent cataractous lenses were significantly higher (254.0 +/- 155 pmo l/mg protein, P < 0.005) than in age-matched, aged (16.1 +/- 8 pmol/mg) or nuclear cataractous lenses (49.0 +/- 26 pmol/mg). Lenses from diabetic indi viduals showed a modest increase (50.3 pmol/mg) compared with age-matched n ormal lenses. HPLC results provided additional evidence that human lenses c ontain argpyrimidine. Western blotting experiments showed consistently stro nger reactions with cataractous lens proteins than those from noncataractou s lenses, and argpyrimidine was found in both crystallin monomers and polym ers. Al crystallins and several cross-linked high-molecular-weight aggregat es reacted with the antibody to argpyrimidine, but a protein of similar to 28 kDa in the alpha -crystallin fraction displaced the greatest immunoreact ivity. CONCLUSIONS. Methylglyoxal modifies arginine within the human lens, and the changes occur at a much higher rate in brunescent lens proteins than in ei ther nuclear cataractous or normal lenses. All crystallins contained argpyr imidine and covalently cross-linked aggregates. This is the first report of immunologic evidence for an arginine modification in the human lens by a p hysiologically important alpha -dicarbonyl compound.