Xanthine

To determine second order rate constants of reactions with O₂^•−, each reactant was individually mixed with 50 μM p-nitro blue tetrazolium (NBT), an established O₂^•− scavenger with a second order rate constant of k_NBT = (5.94 ± 0.5) × 10⁴ M⁻¹ s⁻¹ (Bielski and Richter, 1977 ). The reactions were initiated by adding xanthine/xanthine oxidase as a source of O₂^•−. The ultimate reactions conditions were as follows: 0.1 M Na₄P₂O₇, 100 nM deferoxamine (Sigma), 100 μM xanthine (Sigma), 10 mU xanthine oxidase (Sigma) at pH 8.3. The reaction between O₂^•− and NBT was monitored spectrophotometrically at 560 nm. The second order rate constants of each reactant was determined as a steady-state approximation by evaluating the reaction when r_NBT = r_reactant ≡ k_NBT[NBT][O₂^•−] = k_reacant[reactant][O₂^•−]. Therefore, kreacant = k_NBT[NBT]/[reactant].

Generation of transgenic PRU parasites, was accomplished after harvesting tachyzoites, as described previously [14 (link)]. They were then mixed with 5 μg of CRISPR plasmids and 15μg of the appropriate homologous repair construct as plasmids linearized by restriction digest. Parasites were transfected by electroporation, and allowed to recover on HFF monolayers for 24 h. Positive selection for the (HypoXanthine-Xanthine-Guanine PhosphoRibosyl Transferase (HXGPRT) cassette was done with 25 μg/mL mycophenolic acid (Sigma-Aldrich, St. Louis, MO) supplemented with 50 μg/mL Xanthine (Sigma-Aldrich, St. Louis, MO) [17 (link)]. Negative selection against the HXGPRT cassette was done with 340 μg/mL 6-ThioXanthine [18 (link)].

GO was prepared using a modified Hummers method and purified as described previously.⁶⁸ RGO was produced by heating multilayer GO flakes at 250 C for 30 min in nitrogen flow. Few layer graphene (3–5 layers with nominal lateral dimension of 800 nm) was obtained commercially and characterized. Ascorbic acid, titanium (IV) oxide (TiO₂) nanopowders (<25 nm particle size, >99.5 % trace metal basis), iron (II) sulfate haptahydrate (FeSO₄·7H₂O), reduced GSH, D-mannitol, pC60, xanthine, xanthine oxidase from bovine milk grade IV,2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), thiobarbituric acid (TBA), trichloricacetic acid (TCA), DPPH, 2-deoxy-D-ribose (deoxyribose), sodium dodecyl sulfate (SDS), ABTS, β-Nicotinamide adenine dinucleotide 23-phosphate reduced tetrasodium salt hydrate (NADH), phenazinemethosulfate (PMS), xanthine, xanthine oxidase from bovine milk, and NBT were purchased from Sigma Chemicals Co (St. Louis, MO). Hydrogen peroxide (H₂O₂), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA). 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD). ThioGlo-1 Fluorescent Thiol Reagent is purchased from EMD Millipore Chemical (Darmstadt, Germany), Nanopure water was used throughout.