1994 Volume 43 Issue 1 Pages 51-56
The nitrobluetetrazolium (NBT) method was improved for the spectrophotometric determination of the superoxide ion (O2- ). The proposed method is carried out as follows; pour 0.3 ml of a DMSO solution containing O2- (concentration, <2×10-1 mM) into a 30 ml test tube, in which 0.1 ml of O2--free DMSO was previously added, add 4.6 ml of a 2 mM NBT aqueous solution adjusted to pH 7.4, and finally add 1 ml of a 10 mM MgSO4 aqueous solution to this solution. The solution contains the precipitate of blueformazan formed; the precipitate aggregates by shaking for 45 min at 40°C. Filter the precipitate with a cellulose nitrate membrane filter (pore size of 0.45 μm), and dry it with suction ; dissolve firstly the membrane filter in 2 ml of DMSO, and further dissolve the blueformazan by addition of 8 ml hexamethylphosphoramide; measure the absorbance of this solution at a wavelength of 572 nm. The calibration curve of O2- was linear in the range of 0.2×10-1 to 2.0×10-1 mM. This procedure can be applied to the research of scavengers for O2-.
