Effects of thermal denaturation on protein glycation.
Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.
Seidler
Norbert W
NW
Department of Biochemistry, University of Health Sciences, Kansas City, MO 64106, USA. nseidler@uhs.edu
Yeargans
George S
GS
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002336:Carnosine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D005985:Glyceraldehyde / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006358:Hot Temperature; D066298:In Vitro Techniques; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D011489:Protein Denaturation; D011765:Pyruvaldehyde / Q000494:pharmacology; D013552:Swine
D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D009580:Nitroblue Tetrazolium; D005985:Glyceraldehyde; D011765:Pyruvaldehyde; D002336:Carnosine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D001219:Aspartate Aminotransferases