In-gel peroxidase assay for cytochrome c detection

Peroxidase activity of cytochrome c on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) was detected as previously described (Thomas et al., 1976). Briefly, protein extracts were prepared from 3-days-old biofilms suspensions grown at 18°C or 30°C in Lept or Lept + Mn, as described in the Mn(II) oxidase activity section. Protein samples (150 µg) were prepared for electrophoresis by overnight dialysis at 4°C against 10 mM Tris-HCl, 1 mM EDTA, 20% glycerol and 5 pg ml⁻¹ of pyronin Y and were run on an SDS-PAGE 15% with some changes. Sulfhydryl reducing agents were specifically omitted from the samples. The SDS concentration was 0.1% both in the electrophoresis buffer and the gels. The gels were pre-electrophoresed overnight at 1 mA per gel. The SDS was omitted during gel polymerization and was introduced into the gel by the pre-electrophoresis step. Electrophoresis was performed on gels at 4°C. The current was 1 mA per gel until the sample had completely entered the gel (∼1 h). The current was then increased to 3 mA per gel.

Peroxidase activity of cytochrome c was revealed by staining with a fresh solution of 6.3 mM 3,3′,5,5′-tetramethylbenzidine (TMBZ) in methanol. Immediately before use, 3 parts of the TMBZ solution were mixed with 7 parts of 0.25 M sodium acetate, pH = 5. The gel was immersed in the mixture at room temperature and protected from light with shaking every 10–15 min. After 2 h, H₂O₂ was added to a final concentration of 30 mM. Staining was visible within 3 min and increased by 30 min. At that time, the gel was placed in a solution of isopropanol: 0.25 M sodium acetate, pH = 5, in a 3:7 ratio. This solution was replaced twice with fresh solution to remove precipitated TMBZ (Thomas et al., 1976). For Coomassie brilliant blue staining, SDS-PAGE 15% with 15 µg of total proteins were run. Gels were scanned and ImageJ was used to quantify the intensity of bands. Differential bands were cut-out of the activity gels and mass spectrometry (MS) analyses were performed at the Mass Spectrometry Unit of the Institute of Molecular and Cellular Biology of Rosario (UEM-IBR), Argentina.