Trichloroacetic acid tca

rNg-ACP proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing and native conditions (QIAexpressionist system manual; Qiagen, United Kingdom). Pilot expression trials of rNg-ACP expressed from E. coli BL21(DE3)pLysS-pRSETA-ng-acp demonstrated that the recombinant protein was insoluble and required suspension in 100 mM NaH₂PO₄–10 mM Tris-HCl (pH 8.0) buffer containing 6 M guanidinium chloride (GuHCl) (14 (link)). Protein bound to Ni-NTA resin was eluted using 100 mM NaH₂PO₄–10 mM Tris buffer containing 6 M GuHCl (pH 4.5) and precipitated by adding trichloroacetic acid (TCA; BDH, United Kingdom) to reach a 5% (wt/vol) final concentration, and the precipitate was solubilized in phosphate-buffered saline (PBS) containing 0.5% (wt/vol) SDS. In contrast, rNg-ACP expressed from E. coli BL21(DE3)pLysS-pET22b-ng-acp was soluble and was purified using native conditions, as described previously (15 (link)), with bound protein eluted using 50 mM NaH₂PO₄–300 mM NaCl–250 mM imidazole buffer (pH 8.0) and subsequently dialyzed against PBS (pH 7.4) for 48 h. Samples obtained during the purification procedures were analyzed using 14% (wt/vol) SDS-PAGE as described previously (22 (link)), and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Thermo-Scientific, United Kingdom).

Succinic acid, potassium dihydrogen and mono-hydrogen phosphate, glycine, pyrogallol, hydrogen peroxide, trichloroacetic acid (TCA) and ethylenediaminetetraacetic acid (EDTA) sulphosalicylic acid and thiobarbituric acid (TBA) were purchased from BDH, England. 5,5-dithiobis-2-nitrobenzoic acid (DTNB), dihydrogen phosphate, trichloroacetic acid, carbon tetrachloride, and thiobarbituric acid were obtained from Merck Company, Darmstadt (Germany). Rest all other chemicals including carbon tetrachloride (CCl₄) were from Sigma-Aldrich Company (St. Louis. MO, USA). Thymoquinone (TQ) was naturally derived from the Nigella sativa leaves extract in the present study.