Superoxide dismutase formazan

The evidence that O is a product of the burst has already been summarized. Another direct consequence of the formation of O is the reduction of nitroblue tetrazolium to form an insoluble deposit of blue formazan. The reduction of nitro blue tetrazolium had been observed to occur in PMNs in patients with bacterial infections. In intact cells this appears to be a consequence of the formation of O it fails to occur in PMNs from patients with chronic granulomatous disease which cannot undergo the respiratory burst it is diminished by anaerobiosis and it is inhibitable by superoxide dismutase... 

Ogawa et al. (1997) investigated the formation of H202 and the superoxide radical (02 ) in spinach (Spinacia oleracea) hypocotyls with the use of histochemical stains. Nitroblue tetrazolium (NBT) is used to detect 02 radicals. The colored reaction product formazan was only detected in the vascular tissue of developing spinach hypocotyls if CuZn-superoxide dismutase (CuZn-SOD E.C. 1.15.1.1) was inhibited by DDC... 

Superoxide radicals are another factor in oxidative damage. They can be determined with nitrobluetetrazolium (NBT), which then forms the colourless formazan. When melanoidins scavenge the superoxide radicals, the colour of the NBT persists.490,491 The activity of a glucose-glycine melanoidin on superoxide radicals is equivalent to the effect of 16 units of superoxide dismutase. The effect of the HMM and LMM fractions of this melanoidin is almost the same. The reaction rate constant of the melanoidin was markedly higher than that of ascorbic acid. If this were due to the reductone structures embedded in the melanodin, it is difficult to explain why the reducing power of the melanoidins is only 0.7 that of ascorbic acid.490... 

Nishikimi et al., (1972) developed an assay for superoxide dismutase, using phenazine methosulfate which is structurally related to flavin. In this assay phenazine methosulfate is reduced by NADH, and on reoxidation, O2 was generated. The O2 was detected with nitroblue tetrazolium, and maximum inhibition of blue formazan formation by superoxide dismutase was 95% indicating that 5% of the reduction was due to direct interaction between the nitroblue tetrazolium and the reduced phenazine methosulfate. Addition of around 30 ng copper/zinc superoxide dismutase resulted in 50% inhibition of formazan formation. The assay was also used to locate the enzyme on polyacrylamide gels. 

The nitroblue tetrazolium assay (111) is another indirect method that is used especially for detecting SOD activity on gel electrophoresis. Superoxide radicals are generated by xanthine/xanthine oxidase or by the photoreduction of flavins (typically riboflavin), which oxidize H2O to O2. The gel on which SOD samples have been loaded is then stained with nitroblue tetrazolium chloride. This reagent is reduced by superoxide to the blue-colored formazan. SOD competes with nitroblue tetrazolium and produces colorless zones on the blue gels. This method, which is highly speciflc toward superoxide dismutase, is limited by its low reliability with respect to quantitative determinations. 

Various tetrazolium compounds, introduced into enzyme research by Kuhn and Jerchel (1941) and Lakon (1942), are reduced to scarlet-red (2,3,5-triphenyl-2H-tetrazolium), purplish (neoetrazo-lium) or blue (ditetrazolium) formazans, insoluble in water and soluble in fats. Formazan generation is practically inhibited by superoxide dismutase as well as by catalase (Bromme et al. 1999). When superoxide radical generation via the xanthine-xanthine oxidase system was measured by chemiluminescence an addition of nitroblue tetrazolium immediately abolished lucigenin-enhanced chemiluminescence. Nitroblue tetrazolium did not inhibit xanthine oxidase. 

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