Gels were stained using Coomassie® G-250 Stain (SimplyBlue™ SafeStain, Invitrogen) for detection of proteins. For detection of heme-containing proteins, 3,3′,5,5′-tetramethylbenzidine was used for staining, as described previously

(Thomas et al., 1976). The appropriate fractions from gel filtration were pooled and concentrated by Amicon Ultra-15 Centrifugal Filter Units (Millipore) to ∼400 μL. The concentration of cytochrome c and content of heme was determined by pyridine hemochrome analysis (Berry & Trumpower, 1987). UV-VIS spectra were recorded on Shimadzu UV1601PC spectrophotometer. click here The molecular weight was determined by direct electrospray MS with an LTQ-Orbitrap Velos instrument. The purified protein was desalted, dried and dissolved in 0.1% formic acid in 50 : 50 water : acetonitrile. The MS analysis was performed by Proteomics Core Facility at University of Gothenburg, Sweden. Chlorate reductase was purified as described earlier (Thorell et al., 2003) with the modification that

cells were disrupted using a Bead beater (Biospec products) and that the polyethylene imine precipitation step was omitted. Protein concentration of chlorate reductase was determined by Pierce ®BCA Protein Assay Kit (Thermo Scientific). For kinetic studies, the purified cytochrome c-Id1 was reduced using a slight excess sodium dithionite. A stock solution containing nominally 6 mg dithionite mL−1, 17 mM NaOH and 4 μg mL−1 catalase was prepared using nitrogen-flushed water, and was standardized against horse heart cytochrome c. The reduction of cytochrome c-Id1 see more was monitored spectrophotometrically (Shimadzu UV1601PC; ultra-micro cuvette, Hellma, Sigma-Aldrich Sweden AB, Stockholm). The reaction medium was bis-tris-propane (25 mM, pH 7.2) and the final concentration of cytochrome was varied between 4 and 0.6 μM. Samples were mixed with catalytic amount of chlorate reductase (final concentration Silibinin about 0.14 μM) and dithionite (final concentration 28 μM).

Reactions were initiated by addition of chlorate (final concentration 85 mM) and followed by repeated recordings of spectra at 580–530 nm at 1-min intervals. Purification of the cytochrome c from periplasm using hydrophobic interaction chromatography followed by gel filtration, as described above, resulted in the preparation analyzed in Fig. 1. Fractions from gel filtration were analyzed by SDS-PAGE and stained for protein (Fig. 1a) or heme (Fig. 1b). The fractions denoted by arrows were judged sufficiently pure for further characterization. According to the gel electrophoresis, an apparent molecular weight of 6 kDa was estimated. However, MS analysis results in a value of 9434.7 Da. Analysis of tryptic peptides from in-gel digestion confirms that the purified cytochrome is the target protein described and denoted as the 6-kDa cytochrome c in the previous paper (Bäcklund et al., 2009).