HPLC with chiral stationary phase for separation and kinetics study of aspartic acid epimerization in Peroxiredoxin 2...

Amino acid epimerization, a process of converting _L-amino acids to _D-amino acids, will lead to modification in the protein structure and, subsequently, its biological function. This modification causes no change in protein m/z and may be overlooked during protein analysis. Aspartic Acid Epimerization (AAE) is faster than other amino acids and could be accelerated by free radicals and peroxides. In this work, a novel and site-specific HPLC method using a chiral stationary phase for determining the AAE in the active site model peptide (AP) of Peroxiredoxin 2 has been developed and validated. The developed method showed good linearity (1 - 200 μg/mL) and recoveries of the limit of quantification (LOQ), low, medium, and high concentrations were between 85% and 115%. The Kinetics of AAE in AP were studied using the developed method, and the results showed that when ascorbic acid and Cu²⁺ coexisted, the AP epimerized rapidly. The AAE extent increased with time and was positively correlated with hydrogen peroxide generation.