Redox State Of Erythrocyte Peroxiredoxin 2 During Oxidative Stress And Its Effect On Membrane Binding

Peroxiredoxin;Erythrocyte

Bayer, Simone Birgit ; Winterbourn, Christine ; Hampton, Mark

University of Otago

关键词: Peroxiredoxin;    Erythrocyte;   

Others  :  https://ourarchive.otago.ac.nz/bitstream/10523/5786/1/BayerSimoneB2015PhD.pdf

美国|英语

来源: Otago University Research Archive

【 摘 要 】

Peroxiredoxin 2 (Prx2) is the third most abundant protein in erythrocytes. It is a thiol-specific antioxidant protein that is able to reduce hydroperoxides. Prx2 is a physiologically vital protein, since mice lacking Prx2 develop severe haemolytic anaemia. Much is known about Prx2 structure and activity, and its redox state has been linked to several functions in other cells. However, if and how the redox state of erythrocyte Prx2 is modified during inflammation or blood storage has not been established. Further, Prx2 membrane binding in erythrocytes has been observed frequently, but it is not clear if Prx2 redox state affects membrane binding. The aims of this thesis were to investigate how inflammation, neutrophil activation and blood storage affect Prx2 redox state, and how Prx2 membrane binding is influenced by its redox state.When oxidised, Prx2 is converted to a disulfide-linked homodimer. Non-reducing SDS-PAGE and Western blotting was exploited to measure the redox state of Prx2. Prx2 was oxidised when exposed to hydrogen peroxide (H2O2). Neutrophils generate H2O2 during inflammation, so activated neutrophils were able to oxidise Prx2 in neighbouring erythrocytes. Prx2 oxidation by neutrophil-derived H2O2 was also observed in a mouse model of endotoxaemia, raising the possibility of Prx2 being a useful real-time marker for oxidative stress in inflammatory diseases.For blood transfusions, erythrocytes are stored at 4 °C for up to six weeks. Under standard storage conditions, Prx2 remained mainly reduced during the first three weeks, after which oxidised Prx2 accumulated progressively. While supplementation with dihydrolipoic acid (DHLA) showed some promise in prevention of Prx2 oxidation, storage of erythrocytes in an alternative buffer similar to a recently FDA approved solution with high pH and some buffering capacity delayed the onset on Prx2 oxidation successfully. When the antioxidant capacity of stored erythrocytes was tested with additional H2O2, it was revealed that while Prx2 recycling was impaired, it was not absent. This is likely due to a heterogeneous population of erythrocytes, where older cells may lose their ability to recycle oxidised Prx2. Prx2 could therefore be a useful biomarker for oxidation in stored erythrocytes. Prx2 binds to the erythrocyte membrane, which was previously suggested to depend on Prx2 redox state. In whole erythrocytes, Prx2 membrane binding increased with calcium, and decreased with H2O2, while the redox state of Prx2 was only affected by the latter. Furthermore, when both calcium and H2O2 were added, no Prx2 was lost from the membrane, indicating that two different mechanisms are involved in Prx2 membrane binding. After purification of erythrocyte Prx2, isolation of erythrocyte membranes, and incubation of the membranes with hyperoxidised, reduced and oxidised Prx2, it was established that membrane binding was independent of Prx2 redox state. This indicates that H2O2 acts on another component of erythrocytes to inhibit binding. When oxyhaemoglobin and haemichromes, which are denatured haemoglobin, were added, the haemichromes interfered with Prx2 binding to the membrane. Haemoglobin had no effect. This is consistent with the hypothesis that Prx2 protects the membrane from oxidative damage arising from haemoglobin oxidation, but is displaced during erythrocyte senescence.