Fig. 1.

Tagging Prdx2 does not impede reversible dimerisation upon oxidation, but an N-terminal His-tag affects Prdx2 peroxidase activity. (A) HEK293T cells were transfected with BirA*-Flag-Prdx2 or pCI-His-Prdx2 and BirA*-Flag-Prdx2 transfected cells were additionally treated with 50 μM biotin with for 24 h. Cells were treated with or without 100 μM H₂O₂ for 15 s, followed by 50 mM NEM for 5 min before lysis and then analysed by non-reducing SDS-PAGE with subsequent blotting against Prdx2. The samples in lanes 3, 6, 9 are treated with 50 mM DTT. Beta-actin was used as a loading control. (B) Coupled assay of peroxidase activity. Progress curves for the consumption of NADPH at 340 nm for 0.5 μM pulled down BirA*-Flag-Prdx2, His-Prdx2, and recombinant human Prdx2 in the presence of 500 μM NADPH, 1 μM CgTrxR and 8 μM CgTrx are shown. The reaction was started with the addition of 20 μΜ H₂O₂. Data were normalised for the reaction in the absence of H₂O₂ and fitted with an exponential decay in Prism 9.