Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate
Section snippets
Reagents
All chemicals obtained commercially were of the highest purity grade and were used without additional purification. Pure 2-OH terephthalate was kindly provided by Prof. Lewis Kirschenbaum (University of Rhode Island, Kingston, RI). Potassium phosphate monohydrate, DMSO, water and organic solvents, all HPLC grade, were purchased from Fisher Scientific (Fairlawn, NJ). Terephthalate was obtained from Aldrich (Milwaukee, WI). Superoxide dismutase (SOD), catalase, EDTA (sodium salt), isoniazid,
Results and discussion
Fluorescence methods have been developed to determine, in real time, the 2-OH terephthalate formation directly in a cuvette [26], [27]. In our initial experiments the calibration curve obtained with the 2-OH terephthalate standard (added to pure 20 mM potassium phosphate buffer, pH 7.4) demonstrates high sensitivity i.e. lower limit of 1.0 nM 2-OH terephthalate. However, background fluorescence increased significantly after the addition of components required for microsomal-supported reactions,
Acknowledgements
We thank Professors Gisela Witz and Michael Iba for helpful support and critical comments. The authors acknowledge the kind donation of 2-OH terephthalate by Professor Lewis Kirschenbaum.
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