L. Luers, K. Rysiewski, C. Dumpitak, D. Willbold, E. Birkmann

Reducing sugars or reactive dicarbonyl compounds play a major role in glycation of proteins in vivo. Glycation of proteins is the first step in a non-enzymatic reaction resulting in advanced glycation endproducts (AGEs). AGEs can change the biological activities or even inactivate proteins [1; 2; 3]. AGEs formed by reducing sugars are also able to crosslink proteins leading to insoluble aggregates. Therefore it is important to understand the mechanism of AGE-formation. Here, we systematically analyzed the kinetics of AGE-formation in vitro on bovine serum albumin (BSA) as a model protein by fluorescence and absorption measurements utilizing a microplate reader system. We compared different concentrations of BSA. As AGE inducing agents we applied different concentrations of reducing sugars (D-ribose, D-glucose, D-frutose) and reactive dicarbonyl compounds (methylglyoxal (MG) and glyoxal (GO)). Our results show, that at equimolar ratios, the reaction of MG with BSA leads to the highest fluorescence intensity within days, followed by D-ribose, glyoxal, D-fructose and D-glucose. Both D-fructose and D-glucose showed 8 to 10-fold weaker effect in comparison to MG. Results obtained by fluorescence scans was confirmed by absorption measurements. In summary, this experimental setup enables us to measure the kinetics of AGE formation in a fast and defined way. Furthermore, this system is expandable to measure a plurality of different proteins or AGE-inducing agents at the same time. Citations: [1] K. Arai, S. Maguchi, S. Fujii, H. Ishibashi, K. Oikawa, N. Taniguchi, Glycation and inactivation of human Cu–Zn-superoxide dismutase. Identification of the in vitro glycated sites, J. Biol. Chem. 262 (1987) 16969–16972. [2] R. Blakytny, J.J. Harding, Glycation (non-enzymic glycosylation) inactivates glutathione reductase, Biochem. J. 288 (1992) 303–307. [3] M.M. Heath, K.C. Rixon, J.J. Harding, Glycation-induced inactiva- tion of malate dehydrogenase protection by aspirin and a lens molecular chaperone, alpha-crystallin, Biochim. Biophys. Acta 1315 (1996) 176–184.

Keywords (Optional): 
advanced glycation endproducts (AGEs)
reducing sugars