Experimental Materials Methotrexate, CuCl2 × 6H2O, TSP-d4 (trimethylsilyl propionate), D2O, DNO3, NaOD, and pUC18 plasmid

DNA were obtained from Sigma-Aldrich Co, Germany. NaOH, HCl, and ethylene glycol were purchased from Merck KGaA, Germany. Calibration buffers at pH values 4.01 and 9.21 was received from Mettler-Toledo GmbH, Germany. Potentiometric measurements Potentiometric titrations of MTX and its complexes with Cu(II) in aqueous solution in the presence of 0.1 M KCl were performed at 298 K under argon atmosphere using pH-metric titrations (Metrohm, 905 Titrando). The CO2 free NaOH solution was used as a titrant. The samples were titrated in the pH region 2.0–10.5 using a total volume selleck chemicals llc of 1.5 mL. Changes in pH were monitored with a combined glass–Ag/AgCl electrode (Metrohm, Biotrode) calibrated daily by HCl titrations (Irving et al., 1967). Ligand concentration was 5 × 10−4 M, and metal to ligand molar ratios of 1:1 and 1:4 were used. These data were analyzed using the SUPERQUAD program (Gans 1983). Standard deviations (σ values) quoted were computed by SUPERQUAD and refer to random errors. Nuclear magnetic resonance

(NMR) 1H NMR and 13C NMR measurements were performed on a Bruker AMX-500 instrument (1H: 500 MHz). TSP (trimethylsilyl propanoic acid) was used as an internal standard. Samples were prepared in 500 µl D2O (99.95 %) and the final concentration see more was 10 mM and 40 mM for proton and carbon spectra, respectively. NMR spectra

were recorded for MTX and Cu(II)–MTX system at pD (pH measured by electrode uncorrected for the isotopic effect) value 7.5, which after appropriate correction (Krężel and Bal, 2004) is equal to 7.4. Measurements were made for solutions at five different Cu(II)–MTX molar ratios 1:500 ÷ 5:500. The pD of samples was adjusted by adding small volumes of concentrated DNO3 or NaOD. Infrared spectroscopy (IR) The oxyclozanide room temperature infrared powder spectra were recorded using Bruker IFS-66 FT spectrometer. The scanning range was 4,000–400 cm−1 and the resolution was 2 cm−1. Spectra of MTX alone and the Cu(II)–MTX complex were registered in a transmission mode as KBr pellets. DNA strand break analysis The ability of Cu(II)–MTX complex to induce single- and/or double-strand breaks in the absence or presence of H2O2 was tested with the pUC18 plasmid on 1 % agarose gels containing ethidium bromide. The buffered samples (phosphate buffer, pH 7.4) contained combinations of DNA (25 μg/mL) and the components of investigated systems (metal ion and/or antibiotic, H2O2). Concentrations of each substance are given in figure captions.