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M.D. Linetsky, E.V. Shipova; 2–Guanidinoethyl Mercaptan Derivatives as Potent Inhibitors of Glycation–Mediated Protein Crosslinking . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4120.
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Aged human lenses or cataracts exhibit high molecular weight protein aggregates (HMW) containing non–reducible crosslinks. Some of these crosslinks represent Advanced Glycating Endproducts (AGE) that are derived from the reaction between sugars or ascorbic acid degradation products and Lys or Arg residues of lens proteins. The purpose of this study was to investigate the effect of 2–guanidinoethyl mercaptan derivatives on the formation AGE crosslinks in lens proteins.
2–Guanidinoethyl mercaptan (GEM), Bis–2–Guanidinoethyldisulfide (GEM–D) and S–2–Guanidinoethyl pyridoxamine (GEM–P) were synthesized and their identities were verified by NMR and MS spectra. The relative abilities of glucose (GLC), dehydroascorbic acid (DHA) and methylglyoxal (MGO) to crosslink proteins were measured by a sugar–mediated incorporation of N–alpha–biotinyl–Lys (isoB) into lens proteins followed by SDS–PAGE and 2–D electrophoresis in combination with Western blotting. Sterile solutions of calf lens protein (5.0 mg/ml), sugar (10 mM–0.2 M), isoB (2.0 mM) and inhibitor (2–10 mM) in 0.1 M phosphate buffer containing 0.5 mM DTPA, pH 7.0 were incubated for 7 days (MGO), 14 days (DHA) and 28 days (Glc) at 37ºC. At the end of each incubation the reaction mixtures were dialyzed and the isoB incorporated into protein was determined by a HABA–avidin assay.
Incubation of lens proteins under the described conditions with MGO (10.0 mM), DHA (10.0 mM) and GLC (0.2 M) sugars led to the loss of NH2–groups (0.26 ±0.01 µmol/mg protein) by 34%, 42% and 30%, respectively and to the incorporation of isoB into the proteins by 10.9±0.4 nmol/mg protein for MGO, 11.9±0.6 nmol/mg protein for DHA and 2.5±0.6 nmol/mg protein for GLC, respectively. The presence of GEM at 2.0 mM level in MGO mixtures decreased isoB incorporation into the proteins by 76% and increased the level of NH2 groups to 79% of the original protein NH2 levels and a similar effect has been determined with this inhibitor in DHA and GLC mixtures. The data from SDS–PAGE and 2–D electrophoresis followed by Western blot analysis of these mixtures confirmed these results and showed a complete disappearance of HMW proteins in MGO mixtures and significant decrease in HMW protein aggregates in the mixtures containing DHA and GLC. GEM–P and GEM–D have been shown to possess similar inhibitory properties in terms of AGE–crosslinking in lens proteins.
This study demonstrates that low toxicity GEM derivatives with anti–glycation and antioxidant properties may have a therapeutic application in preventing age and diabetes–dependent human lens protein crosslinking
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