Dissertation:
The 13 C isotope labeling as a method to study the effect of protein and carbonyl diamide admixtures on the formation of Amadori products mgr Martyna Kielmas-Pałach
Supervisor: prof. dr hab. Piotr Stefanowicz
Glycation also know as Maillard reaction is the condensation of the reactive carbonyl group of sugars to lysine, arginine or N-terminal amino group in peptides and proteins. The products of this reaction are Amadori compounds, and intermediate as well as advanced glycation products. These compounds can accumulate in organisms and lead to structural and functional modifications of tissue proteins. These modifications influence the natural process of aging and pathogenesis of some diseases.
The main objective of this dissertation was to analyze the potential effect of a proteinprotein and a protein-urea interactions on the early stage of glycation. The crucial stage of the research was elaboration of model conditions for reaction proceeding in complex systems in vivo . The research was conducted on biologically important proteins: fibrinogen, human serum albumin and α-crystallin.
The study was based on the differential glycation isotopic labeling metod. The investigated protein was mixed with D-glucose with natural isotopic composition and the additional protein or urea. The second sample of protein was mixed with [
13
C
6
]D-glucose only. Then the samples were combined, and after reduction of disulfide bonds subjected to proteolysis. Finally, the products of enzymatic hydrolysis were anylysed using liquid chromatography coupled with the mass spectrometry (LC-MS).
The data analysis for five systems has enabled accurate identification of glycated peptide fragments in model proteins. For the selected lysine residues of target proteins inhibitory effect on glycation was observed and quantified on the basis of the difference in intensities of signals generated for extracted ion chromatograms (EIC) of labeled and unlabeled peptide-derived Amadori products. The effect of inhibition of the reaction was dependent on the position of the lysine residue in the sequence. The obtained results suggests, that the observed glycation inhibition is a result of nospecific interactions of proteins.