Figure 4. Additional Oxidative Protein Damage Assays.

(A) In vitro IR-induced oxidative protein damage. Western blot (W) immunoassay of protein-bound carbonyl groups in D. radiodurans (non-irradiated) cell extract adjusted to 500 μM FeCl₂ and irradiated to the indicated doses. A total of 20 μg of protein extract loaded per lane.

(B) Mn-depleted, radiosensitive D. radiodurans cells [1] are highly susceptible to oxidative protein damage during irradiation. D. radiodurans was grown in defined rich medium without Mn supplementation (no-Mn DRM) [1] to OD₆₀₀ 0.8 and exposed aerobically to 10 kGy. A total of 20 μg of protein extract loaded per lane.

(C) Decreased survival of D. radiodurans irradiated at pH 10.5 correlates with oxidative protein damage. D. radiodurans was grown to OD₆₀₀ approximately 0.9 in TGY (pH 7), adjusted to pH 10.5, and exposed aerobically to the indicated doses. A total of 20 μg of protein extract loaded per lane.

(D) P. putida proteins are similarly susceptible to oxidative protein damage when cells are irradiated anaerobically (+Ar) or aerobically (+O₂). P. putida was grown to OD₆₀₀ approximately 0.9 in TGY, purged with ultra-high purity Ar, and irradiated in sealed tubes to 4 kGy.

Values for intracellular Mn/Fe concentration ratios and D₁₀ at the bottom of (B), (C), and (D), as reported previously [1]. A total of 20 μg of protein extract loaded per lane.

C, Coomassie-stained polyacrylamide denaturing gel; M, mixture of artificial IgG-binding protein standards; O, oxidized protein standards; S, wide-range protein standards; +, DNPH treated;−, DNPH untreated.