4 4 dithiodipyridine

Strains ΔPorNRgpB665sXa6H and ΔPorNRgpB662iXa6H were grown to a stationary phase (OD600 = 1.5) and bacterial cells collected by centrifugation (8,000 × g, 30 min) from 8 L culture. Cells were washed and resuspended in Ni²⁺-Sepharose binding buffer (20 mM sodium phosphate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4, supplemented with 0.02% NaN₃ and 1.5 mM 4,4-dithiodipyridine (Sigma-Aldrich)) and lysed by sonication. Insoluble cell debris were removed by ultracentrifugation (100,000 × g, 60 min, 4 °C) and clarified supernatant was applied onto a column of pre-equilibrated Ni²⁺-Sepharose 6 Fast Flow matrix (1.5 ml) (GE Healthcare). After extensive washing, bound proteins were eluted with binding buffer supplemented with 500 mM imidazole. Final purification of proRgpB variants was accomplished by size exclusion chromatography on Superdex 75 column equilibrated with 20 mM Tris, 50 mM NaCl, 5 mM CaCl₂, 0.02% NaN_3, pH 7.5 using the ÄCTA Purifier system. Fractions containing proRgpB were pooled together, concentrated by ultrafiltration (30 kDa cut-off membrane) and dialyzed against 20 mM BisTris, 150 mm NaCl, 5 mM CaCl₂, pH 6.8 with 0.02% NaN₃. Protein concentration of the final samples was determined by BCA Assay (Sigma, St Louis, MO, USA).

All commercially available reagents and solvents were used as received without further purification. ZrOCl₂ and D₂O were bought from Energy Chemical. Fumaric acid and FeCl₃·6H₂O was purchased from Alfa Aesar. Zn(NO₃)₂·6H₂O, Co(NO₃)₂·6H₂O, Cu(NO₃)₂·3H₂O, 2-methylimidazole, K₂PtCl₄, polyvinyl pyrrolidone, terephthalic acid, dimethyl sulfoxide-d6 (DMSO‑d6), 2,6-dimethylnitrobenzene, 4,4’-dithiodipyridine and biphenyl-4,4’-dicarboxylate were bought from Sigma Aldrich. ZrCl₄ was purchased from Acros Organics.

4-Maleimido-2,2,6,6,-tetramethylpiperidine-1-oxyl (MSL) and 4,4′-dithiodipyridine were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were analytical grade products from POCh (Gliwice, Poland).

Gelatinase activity in placental leukocyte supernatants was evaluated using a specific substrate (Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC2H5, Calbiochem, San Diego, CA), according to Weingarten and Feder [20 (link)]. Supernatants (1.0 μg protein) in a solution containing 0.3 mM substrate, 50 mM HEPES (pH 7.4), and 1.0 mM 4,4′-dithiodipyridine (both from Sigma-Aldrich, St. Louis, MO), were incubated in triplicates for 3 h at 25°C. The 4,4′-dithiodipyridine reacts with the mercaptan hydrolysis fragment to form products that were measured at 324 nm, using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA).