SPECTROPHOTOMETRIC DETERMINATION OF SUPER OXIDE ION ON THE NITROBLUETETRAZOLIUM METHOD

The nitrobluetetrazolium (NBT) method was improved for the spectrophot ometric 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 X 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 pre cipitate of blueformazan formed; the precipitate aggregates by shaking for 45 min at 40-degrees-C. Filter the precipitate with a cellulose n itrate membrane filter (pore size of 0.45 mum), and dry it with suctio n; dissolve firstly the membrane filter in 2 ml of DMSO, and further d issolve 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 X 10(-1) to 2 .0 X 10(-1) mM. This procedure can be applied to the research of scave ngers for O2.