Figure 5. Effects of H₂O₂ exposure on the T. bernacchii 26S proteasome.

(A) Native-PAGE of T. bernacchii 26S proteasome complex after treatment with H₂O₂. Purified 26S proteasome was exposed to increasing H₂O₂ concentrations for 24 h at 37°C and the bands were visualized by Coomassie-blue stained. The experimental conditions for H₂O₂ treatment and native-PAGE are described in Materials and Methods section. (B, left panel) Unoxidized and oxidized BSA were incubated with 26S proteasome at 37°C for the indicated periods; (B, right panel) Unoxidized and oxidized BSA were incubated with H₂O₂-treated 26S proteasome at 37°C for the indicated periods. All the reaction mixtures were subjected to SDS/PAGE. (C) SDS/PAGE data are expressed as percentage density of BSA at the indicated incubation times compared with time 0, and were obtained by densitometric analysis with ChemiDoc XRS and Quantity One software. Oxidized BSA levels after incubation with 26S and H₂O₂-treated 26S are indicated by diamonds and squares respectively. Unoxidized BSA incubated with 26S and H₂O₂-treated 26S are indicated by triangles and circles respectively. The experiments were performed in duplicate on two different protein preparations, and the average of the relative intensities of measurements, performed in triplicate, are expressed as means ± standard deviations.