Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol.

A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of SOD activity is performed at pH 8.8, 37 degrees C, in 50 mM air-saturated 2-amino-2-methyl-1,3- propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT-01050 is stabilized in stock solution acidified at pH 1.5. The SOD activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One SOD activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied SODs. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of SOD activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known SODs such as Cu/Zn-, Mn-, or Fe-SOD.