Abstract
Abstract: :
Purpose: The glycation of calf lens proteins (CLP) by ascorbic acid produces yellow chromophores and fluorophores, which are chromatographically very similar to those isolated from aged human and cataract lens proteins. In the work presented here our aim was to determine whether other glycating agents could also produce chromophores and fluorophores 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 digested with proteolytic enzymes. The AGE-containing peak was isolated from a Bio-Gel P-2 column and subjected to RP-HPLC on a Prodigy column to fractionate the chromophores. Similarly, Nα-t-Boc-Lys was reacted with several glycating agents and analyzed by RP-HPLC. Several inhibitors were employed at a concentration of 10 – 50 mM to confirm that the peaks were due to AGE formation. Results:(1). The reactivity of the various glycating agents for the formation of yellow chromophores and fluorophores with lens proteins were: MGO>L-ascorbic acid>L-erythrulose>D-ribose>D-glucose. (2). Ascorbic acid and L-erythrulose, which is a breakdown product of ascorbic acid, produced similar chromophores and fluorophores when analyzed by RP-HPLC. Ribose produced only a few fluorescent peaks similar to ascorbate and erythrulose, whereas glucose was inactive. MGO gave the highest level of AGE products, however, only a single major peak was seen, which was identified as argpyrimidine. (3). Aminoguanidine, semicarbazide and cyanoborohydride caused a 12, 80 and 64% inhibition of chromophore formation respectively. (4). With the model system, HOP-Lys was confirmed as a major product with L-ascorbic acid, dehydro-L-ascorbic acid (DHA) and L-erythrulose, but not with glucose, fructose or MGO. Conclusions: The yellow chromophores and fluorophores formed from ascorbic acid, DHA and L-erythrulose were almost completely identical. The other carbonyl compounds did not generate the same chromophores and fluorophores, suggesting that the AGEs, which accumulate with age in human lens arise from ascorbic acid oxidation products.
Keywords: protein modifications-post translational • age-related macular degeneration • oxidation/oxidative or free radical damage