Bovine erythrocyte superoxide dismutase sod

Doxorubicin hydrochloride and mitomycin C were purchased from commercial sources. Menogaril, 5-iminodaunorubicin, and mitoxantrone were supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD. 4-demethoxydaunorubicin, 4′-epidoxorubicin, and 4′-deoxydoxorubicin were a gift of Farmitalia Carlo Erba, Milan, Italy. Menadione (2-methyl-1,4-naphthoquinone, sodium bisulfite salt), cytochrome c (type VI from horse heart), dimethyl sulfoxide (DMS0), diethylenetriaminepentaacetic acid (DTPA), hyaluronidase (type I-S), and bovine erythrocyte superoxide dismutase (SOD, 2900 units/mg) were from Sigma-Aldrich Chemical Co., St. Louis, MO. Prior to these experiments, cytochrome c was acetylated to eliminate the effects of potential exogenous oxidizing or reducing species present in our reaction mixtures [16 (link)]. Collagenase (type II) was purchased from Cooper Biomedical Inc., Freehold, NJ. Metrizamide (type AN 6300) of analytical grade was from Accurate Chemical and Scientific Corp., Westbury, NY. Catalase of analytical grade (65,000 units/mg) was purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN, and was devoid of SOD activity when assayed by the method of McCord and Fridovich [17 (link)]. Only glass distilled, deionized water was used in these studies.

Superoxide anion (O2⁻) was produced by the xanthine/xanthine oxidase system and hydroxyl radical (OH) was generated from decomposition of H₂O₂ by ferrous ions. Both reactions were carried out in the presence of a spin trap 5,5-dimethylpyrroline-N-oxide (DMPO). ASCOP solution was added at concentrations ranging respectively from 0.025 to 0.50 µg mL⁻¹ for superoxide anion assay and 0.0025 to 0.05 µg mL⁻¹ for hydroxyl radical assay. Electron Spin Resonance (ESR) spectra were recorded at room temperature using a Bruker ECS 106 spectrometer. The scavenging activity of ASCOP was evaluated by the decrease in the ESR signal expressed as arbitrary units. Scavenging activities were calibrated using the bovine erythrocyte superoxide dismutase (SOD, Sigma-Aldrich) as standard for superoxide anion, and benzoic acid as reference scavenging substance for hydroxyl radical. All ESR experiments were performed five times for each point and data are expressed as means ± SD.