| dc.description.abstract | The most abundant intracellular protein in erythrocytes is haemoglobin (Hb) and the third most abundant protein is peroxiredoxin 2 (Prx2). Prx2 exists in both dimeric and decameric form; its dimeric form has two cysteines (Cys) redox active sites. The presence of the two redox-active Cys sites means that Prx2 possesses free thiols, which can react with reactive nitrogen species to form S-nitrosothiols. The function of Prx2 is not completely understood, although it is thought to be an antioxidant by virtue of its peroxidase activity. Prx2 may play a central role in protecting Hb against oxidative damage and mediating some of the key functions of Hb. Nitric oxide (NO) exerts numerous important physiological functions in biological systems including vasodilation in humans. Because of the short-lived nature of NO, S-nitrosothiols (RSNOs) are believed to act as stable NO carriers. This study hypothesises a model highlighting possible interactions between NO and Hb, and the HbNO complex and Prx2. In this model, NO is produced upon nitrite reduction by deoxyHb, NO equivalents are transferred to the Cys residues of Prx2 forming S-nitrosoperoxiredoxin (Prx2-SNO). The proximity of Prx2 to Hb promotes S-nitrosothiol formation in Prx2 and avoids the scavenging of NO by Hb itself. This study also describes the purification of the native hPrx2, and the over-expression and purification of hPrx2 in various forms, as well as the subsequent crystallisation and X-ray diffraction studies. The calibrated gel filtration column confirmed that hPrx2 is mainly produced in the decameric form. Purification of native hPrx2 isolated from human red blood cells (RBCs) was successfully achieved using FFQ Sepharose and gel filtration chromatography (Superdex 200). The recombinant hPrx2 DNA cloned in pET28q was transformed into a competent E.coli Rosetta Gami 2 cell line. The purified recombinant hPrx2 protein was achieved using nickel affinity and gel filtration column chromatography. S-nitrosated Prx2 (Prx2-SNO) was achieved by incubation of Prx2 with S-nitrosoglutathione (GSNO) under differing experimental conditions. The mixing of hPrx2 and Hb in various molar ratios, demonstrated a significant specific protein-protein interaction between a Prx2 decamer with an Hb tetramer. The complex binding results from size exclusion chromatography showed that decameric Prx2 bound to Hb in a 1:1 ratio. This study also describes the post-modification of hPrx2 cysteine residues, and the nitration of tyrosine residues attained by the incubation of protein with GSNO. The incubation of the recombinant hPrx2 protein with GSNO altered the oligomeric state of hPrx2 into a dimer formation. The gas-phase chemiluminescence, gel filtration chromatography, Saville assay, Western-blot, UV-vis spectrophotometry and LC-MS/MS, confirmed the post-modifications of the protein residues. This research reports the purification and characterization of the stable; native hPrx2, recombinant hPrx2, hPrx2-SNO, and SNO-Hb proteins necessary to perform the hypothesised interactions, to allow us to understand the transnitrosation process that could occur between hPrx2 and Hb. The study also describes the crystallisation conditions for hPrx2-Hb complex and the co-crystallisation conditions for hPrx2-SNO and [hPrx2-SNO]Hb complex | en_GB |
