Research Paper Volume 1, Issue 2 pp 254—265

Figure 3. Functional characterization of the p66Shc/Prx1 interaction. (a) The dimer is the peroxidase-active form of Prx1. Changes in fluorescence of 10 μM H₂DFFDA were recorded after addition of 0.005 % H₂O₂ in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (b) p66CH2CB has an additional copper-independent activity which is inhibited by Prx1. Changes in fluorescence of 10 μM H₂DFFDA were recorded after addition of 20 μM p66CH2CB and 0.005 % H₂O₂ in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (c) p66CH2CB and Prx1 perform a disulfide exchange reaction with each other. 10 μg p66CH2CB were incubated with Prx1 for 1 hour at room temperature and subjected to non-reducing SDS-PAGE. Reduced p66CH2CB is formed, and the reduced Prx1 is concurrently oxidized. (d) Trx1 does not prevent formation of the major p66CH2CB/Prx1 complex, indicating separate binding sites for Prx1 and Trx1. 15 μg p66CH2CB were incubated with 30 μg decameric Prx1-Cys52Ser and different amounts of Trx1 (5/10/20 μg) in the presence of 3 mM EDTA and subjected to BN-PAGE.