Protein Carbonylation

The bacteria were grown as described above. Aliquots of suspended cells were exposed to 1.5, 7.5, 10, and 15 kGy or incubated with PBS containing formaldehyde (0.1, 0.2, 1, 2, and 5%) for 2 h at 37 °C. Total bacterial protein samples were sonicated using a TECAN sonicator (TECAN, Osaka, Japan) for 5 min on ice. The samples were then centrifuged at 10,000× g for 15 min at 4 °C. Protein concentration and purity were determined by measuring the absorbance at 280 and 260 nm. Protein carbonylation was measured using a protein carbonyl colorimetric assay kit (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Briefly, 200 µL of the lysate was mixed with 800 µL 2,4-dinitrophenylhydrazine and 800 µL of 2.5 M HCl. The proteins were then precipitated using 20% trichloroacetic acid (TCA) at 4 °C for 5 min. After centrifugation, the pellet was washed with 1 mL of an ethanol and ethyl acetate mixture (1:1, v/v) and resuspended in 500 µL of guanidine hydrochloride. The optical density (OD) of the pellet was measured at 360–385 nm using a BIOTEX microplate reader.

Skin biopsies were prepared through initial washing with physiological solution (0.9% NaCl). First, 0.5 g of the skin biopsies were subjected to the desired treatment (control or Fenton reagent applied topically for 30 min each), followed by rinsing with distilled water. Subsequently, the samples were snap-freezed in liquid N₂. The samples were then homogenized with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40) comprising 1% (v/v) protease and phosphatase inhibitor (v/v) (three times, 1 min each), followed by sequential centrifugations at 8000 rpm (30 min, 1 time) and 14,000 rpm (45 min, 2 times). The supernatant was collected and quantified with a Pierce BCA protein estimation kit (Thermo Fisher Scientific, Paisley, UK). The detailed sample preparation procedure is presented in Figure 10. Protein samples for Western blotting were prepared with SDS Laemmli sample buffer. The prepared samples were then subjected to electrophoresis and immunoblotting analysis using anti-MDA antibody. For immunoblotting, 2 biological replicates were performed for each measurement.
To detect protein carbonyl formation, the collected protein fractions were subjected to derivatization. Carbonyl groups present in the protein side chains were derivatized with 2,4 dinitrophenylhydrazine (DNPH), leading to the formation of stable 2,4 dinitrophenylhydrazone (DNP) derivative, which involves the addition of an equal volume of protein and 12% SDS (final concentration at 6%) and subsequent addition of 1X DNPH solution (50 mM solution in 50% sulphuric acid). The mixture was incubated at RT for 30 min and the reactions were neutralized with 2 M Tris base and 30% glycerol (0.75× v/v of DNPH solution). The resulting protein fractions were centrifuged at 14,000 rpm for 10 min and the supernatants were loaded onto SDS gels for immunoblotting with an anti-DNP antibody.
Whole cell homogenates (10 μg/lane), processed on 10% SDS gel, were then transferred to blotting membranes (nitrocellulose) using a Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA, USA). The membranes were blocked (BSA in phosphate buffered saline, pH 7.4, containing 0.1% Tween 20) overnight at 4 °C. The blocked membranes were probed for 2 h with an anti-MDA antibody at RT. After 4 cycles of washing with PBST and incubation for 1 h at room temperature with HRP-conjugated anti-rabbit secondary antibody (dilution 1:10,000) and subsequent washing [PBST, 5× (5 min each)], the immunocomplexes were visualized utilizing Immobilon Western Chemiluminescent HRP Substrate (Sigma Aldrich, GmbH, Mannheim, Germany) and imaged using an Amersham 600 imager (GE Healthcare, Amersham, UK). Densitometry analysis of the blots obtained was generated using Image J 1.53t [public domain software (Bethesda, MD, USA) provided by the National Institute of Mental Health, United States].