Direct measurement of superoxide anion produced in biological systems by ESR spectrometry: a pH-jump method.
For direct measurement of the ESR signal of superoxide anion (O2-) produced in biological samples, O2- generated at a physiological pH was trapped in alkaline media instead of by a rapid freezing method, and then its signal was measured by ESR spectroscopy at 77 K. A reaction mixture for O2- generation, such as xanthine oxidase-xanthine and neutrophils, was incubated at a physiological pH (pH 7.0-7.5) for a suitable reaction period (30s), then an aliquot (300 microliters) was pipetted out and squirted into 600 microliters of 0.5 M NaOH to stabilize O2- (pH-jump). The alkaline mixture was promptly introduced into an ESR tube and frozen by dipping the tube directly into a cooling liquid. A typical signal of O2- was detected by ESR spectroscopy and the amount of trapped O2- was measured quantitatively at 77 K. The back reaction of O2- generation from H2O2 was negligible in 0.5 M NaOH. To avoid any artificial spectrum due to autoxidation of biological samples by the pH-jump procedure, the background spectrum should be subtracted from the obtained spectrum. This pH-jump method should be widely available for direct demonstration of O2- production in biological systems at physiological pH, because an advantage of this method is the simple operation for trapping O2- without the use of any rapid-mixing apparatus as compared with the rapid freezing method.
Fujii
H
H
Tokyo Metropolitan Institute of Medical Science.
Kakinuma
K
K
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D012972:Sodium Hydroxide / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013552:Swine; D014969:Xanthine Oxidase / Q000378:metabolism
D013481:Superoxides; D012972:Sodium Hydroxide; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010100:Oxygen