Figure 5
Protection of DNA and peroxidase activity. (A) Metal catalyzed oxidation (MCO) assay. The pBSK DNA (1 μg, lane 1) was subjected to oxidative damage using a MCO reaction (5 mM DTT + 3 μM Fe3+) to generate ROS in absence (lane 2) or in presence of BSA (lane 3) or purified Alr4641 (lane 4). The integrity of DNA was assessed by electrophoresis on a 1% agarose gel followed by staining with ethidium bromide. (B) Peroxidase activity. Relative rates of decomposition of H2O2 by the purified Alr4641 protein using various electron donors: GSH, DTT and TrxA. (C) Peroxidase activity of Alr4641 cysteine mutants. Decomposition of H2O2 by Alr4641C56S or Alr4641C178S or Alr4641 was monitored with 5 μM TrxA as reducing agent at different intervals of time as indicated in the figure. H2O2 was monitored as described in the Methods section. (D) The NTRC protein from Anabaena PCC7120 was over-expressed in E. coli and purified by affinity chromatography as described in the Methods section. After electrophoresis the proteins were visualized by staining with CBB. Lane 1, mol. mass marker and lane 2, purified NTRC protein (5 μg). (E) Surface plasmon resonance analysis. The Alr4641 protein was immobilized on bare gold chip utilizing the EDC-NHS chemistry (Autolab ESPIRIT User manual SPR). Different concentrations of NTRC (as indicated in the figure) were injected over Alr4641 and the response was monitored for 300 s. (F) Peroxidase activity of Alr4641, in the presence of NTRC, was monitored at different concentrations of H2O2 as indicated in the figure.