The synthesis and analysis of S-nitorsylated paraoxonase 1

Post-translational modification (PTM) of proteins plays a crucial role in health and disease by affecting numerous aspects of protein structure, function, stability and subcellular localization. Protein S-nitrosylation is one type of PTM that involves the covalent modification of cysteine sulfhydryl groups with nitric oxide (NO) and has a regulatory impact similar to phosphorylation. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDL's antiatherogenic properties. The enzyme contains a free thiol group at Cys-284 which can also be modified covalently. As part of our effort to study the effect of PTMs on PON1 activities and properties and its implication for cardiovascular disease, we examined PON1's ability to undergo S-nitrosylation on its free Cys-284. Recombinant (re) PON1 was trans-S-nitrosylated by several NO donors, glutathione-NO (GSNO) was found to be the most effective. The S-nitrosylated rePON1 was analyzed by Q-TOF LC/MS and by Saville-Griess assay: the two analytical methods revealed closely similar results. rePON1 was also nitrosylated by nitrosylated human serum albumin (HSA-NO) via protein-protein trans-nitrosylation. HSA-NO transferred an NO group to rePON1 much more efficiently than GSNO with the formation of a higher than 70% rePON-NO when incubated with a 40-fold excess of a HSA-NO/HSA mixture. RePON1-NO was relatively stable: storage for 3 days at 37 °C resulted in only 25% decomposition. This is the first report of PON1's S-nitrosylation via GSNO and HSA-NO.