Characterization of Lens Protein Modifications by Ascorbic Acid and Other Carbonyl Agents With 2–D LC–MS MAP

Abstract

Abstract: : Purpose: The glycation of calf lens proteins (CLP) by ascorbic acid produces yellow chromophores and fluorophores, which are chromatographically and chemically identical to those isolated from aged human and cataract lens proteins. In the work presented here our aim was to determine using 2–D LC–MS MAP analysis whether other glycating agents could also produce chromophores common to those in human lens. Methods: CLP (10 mg/ml) were incubated with either 200 mM glucose or 20 mM ascorbate, ribose, erythrulose or methyl glyoxal (MGO) at 37 ^oC in Chelex–treated phosphate buffer for 4 weeks. Each reaction mixture was dialyzed and the glycated proteins were digested with proteolytic enzymes. The AGE–containing peaks were isolated from a small Bio–Gel P–2 column and subjected to an in–line LC–mass spectrometer equipped with an electrospray interface. Several inhibitors were employed at a concentration of 10 – 50 mM to confirm that the peaks were due to glycation. All the LC–MS data were analyzed using 2–D MAP technique. Results: (a). Bio–Gel P–2 profiles were similar for all the glycated CLP digests, but the reactivity of the various glycating agents for the formation of yellow chromophores with lens proteins were: MGO>L–ascorbic acid>L–erythrulose>D–ribose>D–glucose. (b). Ascorbic acid and L–erythrulose, which is a breakdown product of oxidized ascorbic acid, produced identical modifications confirmed by LC–MS MAP analysis. Glucose, ribose and MGO generated several modifications same to ascorbate and erythrulose, but in much lower levels. (c). Aminoguanidine and cyanoborohydride inhibited almost 80% of the glycation reactions. Conclusions: The modifications formed from ascorbic acid and L–erythrulose was identical. The other carbonyl compounds generated several modifications same as ascorbic acid but in much lower concentrations, suggesting that the AGEs, which accumulate with age in human lens arise mainly from ascorbic acid oxidation products.

Keywords: protein modifications-post translational • aging • cataract