Analysis and Chemistry of Novel Protein Oxidation Markers in Vivo

Abstract

Proteins continually undergo spontaneous oxidation reactions, which lead to changes in structure and function. The quantitative assessment of protein oxidation adducts provides information on the level of exposure to reactive precursor compounds with a high oxidizing potential and reactive oxygen species (ROS). In the present work, we introduce N-(2-hydroxyethyl)lysine as a novel marker based on the ratio of glycolaldehyde and its oxidized form glyoxal. The high analytical potential was proven with a first set of patients undergoing hemodialysis versus healthy controls, in comparison with well-established parameters for oxidative stress. In vitro experiments with N- t-BOC-lysine and N- t-BOC-arginine enlightened the mechanistic relationship of glycolaldehyde and glyoxal. Oxidation was strongly dependent on the catalytic action of the ε-amino moiety of lysine. Investigations on the formation of N-carboxymethyl lysine revealed glycolaldehyde-imine as the more reactive precursor, even though an additional oxidative step is required. As a result, a novel and very effective alternative mechanism was unraveled.

Comprehensive analysis of posttranslational protein modifications in aging of subcellular compartments

“…Here, the values for MG-H are given as a sum for the two isomeric forms MG-H1 and MG-H3. The higher values of MG-H compared to CEL measured in all subcellular fractions is in line with the higher reactivity of the guanidine function of arginine versus the N 6 -amino function of lysine towards α-dicarbonyl compounds 8 . In contrast, the glyoxal specific hydroimidazolinone G-H3 is an artefact of acid protein hydrolysis but has been shown to be a useful quantitative tool for glyoxal-dihydroxyimidazolidine and the AGE N 7 -carboxymethyl arginine 41 .…”

“…In contrast to GALA and glyoxal arginine modifications it has to be considered, that there are major alternative pathways for CML formation. Notably, the oxidative fragmentation of the Amadori product which makes CML also a parameter of oxidative stress 8 . In total, AGEs were excellent markers of aging in all subcellular compartments.…”

“…In order to prevent artefact formation proteins were reduced by NaBD 4 prior to acid and enzymatic hydrolysis as described previously 8 . The average efficiency of enzymatic hydrolysis was about 90%.…”

“…In contrast to the PTMs described above, a plethora of modifications is formed non-enzymatically. Oxidative stress is a well-known mechanism leading to PTMs which are closely linked to inflammation and impaired proteostasis 7,8 . A long-term research field is the formation of advanced glycation endproducts (AGEs), which are established markers of aging in many extra-and intracellular tissues, e.g., in eye-lens proteins 9 .…”

Advanced Glycation End-Products (AGEs) and Their Soluble Receptor (sRAGE) in Women Suffering from Systemic Lupus Erythematosus (SLE)

“…Subsequently, numerous reactions may occur, such as rearrangement, cross-linking, cyclization, isomerization, and cleavage [ 18 , 19 ]. Oxidative conditions and carbonyl stress promote AGEs formation [ 20 ]. Various AGEs are found in body fluids, cells, and tissues of human subjects, including carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, and many more [ 18 ].…”