Figure 7.

Hyperoxidation of wild-type and mutants Tsa1 with H₂O₂ and tBOOH in S. cerevisiae. (a–c) Tsa1 is more reactive toward organic peroxides than with H₂O₂ in vivo. Tsa1 hyperoxidation in cells exposed to H₂O₂, tBOOH, and CuOOH, during 5 min at the indicated concentrations. Thiols were derivatized by N-ethylmaleimide (NEM) or mPEG, as indicated, after reduction with dithiothreitol (DTT), as described in methods. (d–f) Comparison of the H₂O₂ reactivity of Prx1, Tsa1, and Tsa1^A177S A178D in vivo. Thiols were derivatized by NEM or mPEG, as indicated, after reduction with DTT, as described in methods, using cell lysates of Δtsa1 expressing human Prx1, Tsa1, or Tsa1^A177S A178D and exposed to H₂O₂ at the indicated concentration. (a, d) Western blot of reduced (−SH) (2 × mPEG) and hyperoxidized (−SO₂H) (1 × mPEG) forms of Tsa1 (indicated by black arrows), revealed with an anti-Prx1 antibody. (b, e) Western blot of the Prx SO₂H form using a Prx anti-SO_2/3 antibody and the cell lysates used in (a) and (c), respectively. (c, f) Quantification of the degree of oxidation (SO₂H/SH + SO₂H) vs peroxide concentration.