Nitroblue tetrazolium assay

Fig. 3.12. Schematic representation of the procedure for the nitroblue tetrazolium assay...

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. 

All these assays can also be used to determine the activity of MnSOD and FeSOD. It should be noted that the nitroblue tetrazolium assay (111) allows differentiation of MnSOD, FeSOD, and Cu2Zn2 SOD. Indeed, these enz3unes have different molecular weights and they run at different rates on acrylamide gel. In a mixture of the three proteins it is possible to resolve three spots representing Mn, Cu2Zn2, and FeSOD. 

Using the nitroblue tetrazolium assay, Cu(tyr)2 and Cu(lys)2 also show a remarkable SOD-activity However, the rate constants are three orders of magnitude lower than those calculated from the data of pulse radiolytic measurements. Intriguingly, adrenochrome formation, which is used as an indicator of superoxide is accelerated by low molecular mass copper chelates The inhibition of adrenochrome... 

Early methods of superoxide detection are well known and described in many books and reviews. They include cytochrome c reduction, nitroblue tetrazolium reduction, spin trapping, etc. (see, for example, Ref. [1]). The most efficient assays are based on the ability of superoxide to reduce some compounds by one-electron transfer mechanism because such processes (Reaction (1)) proceed with high rates [2] ... 

Stopped-flow measurements with superoxide in aqueous solution at physiological pH are not possible due to its fast self-dismutation under these conditions. Therefore, the indirect assays such as McCord-Fridovich, adrenalin and nitroblue tetrazolium (NET) assays are widely used in the literature, not only for qualitative but also for quantitative detection of SOD activity of small molecular weight mimetics 52). Not going into details, we just want to stress that the indirect assays have very poor even qualitative reliability, since they can demonstrate the SOD activity of the complexes which does not react with superoxide at all. It has been reported in the literature that this is caused by the interference of hydrogen peroxide 29). We have observed that the direct reaction between complexes and indicator... 

FIGURE 3.2 Effect of retinoic acid (RA) and various lipids on cell differentiation of HL-60 cells (Tochisawa et al., 1997). HL-60 cells (5xl04) were incubated with the individual lipids for 24h, then RA was added followed by incubation for additional 72h. Nitroblue tetrazolium (NBT) reducing activity in HL-60 cells was assayed. Data shown as mean value SD. p<0.01 versus control. p<0.01 versus RA. 

A superoxide dismutase activity had been reported for the Fe-EDTA complex in contrast with the inactivity of the Cu-EDTA complex. It was shown, on the contrary, that Fe-EDTA, instead of catalysing the dismutation of OJ, interferes with the reduction of nitroblue tetrazolium and of Fe(III)-cytochrome c in the assays of the dismutase activity... 

The SOD activity is directly determined by following the decay of 05 or indirectly by competition between a monitoring molecule of 05 and SOD for 05. Cytochrome c, nitroblue tetrazolium, tetranitromethane and epinephrine have been used as the monitoring molecules of 05, and their reaction products are spectrophotometrically followed. Since the indirect methods do not require special apparatus, they have been widely used for the assay of SOD. For elucidation of the reaction mechanism, however, the direct methods using special apparatus are indispensable. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

It should be mentioned that most natural aldolase enzymes can also be assayed using enzyme-coupled systems relaying the reaction to a redox process with NAD. The formation of NADH by active microbial colonies in expression libraries of mutant enzymes was detected colorimetrically in agar plates using phenazine methosulfate and nitroblue tetrazolium, which forms an insoluble precipitate. The assay was used by Williams et al. [14] and Woodhall et al. [15] for evolving sialic acid aldolases to accept non-natural aldehyde acceptors. 

Substrate to give a soluble product (plate assays) 0.1% / -Nitrophenyl phosphate in 10 mAfdiethanolamine, pH 9.5, containing 0.5 mAfMgClg. Substrate to give an insoluble product (Western blots) NBT stock— 5% nitroblue tetrazolium in 70% dimethyl formamide. BCIP stock—5% disodium bromochloroindolyl phosphate in dimethyl formamide. Alkaline phosphatase buffer—100 mAf diethanolamine, pH 9.5, containing 100 mAfNaCl and 5 m AfMgClj. Just before use, add 66 lL of NBT stock solution to 10 mL of alkaline phosphatase buffer, mix well, and add 33 pL of BCIP stock soludon. 

Fe" " forms a colored complex with xylenol orange that can be read at 560 nm (Jones et al., 1995). Superoxide radical generation can be estimated by nitroblue-tetrazolium reduction assay (Libon et al., 1993). 

A method for the estimation of serum glycosylprotein, based on the ability of ketoamines (fructosamines) to reduce nitroblue tetrazolium at pH 10.8, was reported in 1982 04). More recently, an automated assay (75 samples per hour) has been described, with excellent precision (coefficient of variation 2%) and low cost (Dl). 

In 1993 Weiss, Riley, and co-workers reported a study on purported SOD mimics by stopped-flow UV-vis spectroscopy (428) in which they assessed reactivity by following the decay of the superoxide absorption at 245 nm. Two of the earlier techniques that had been used to assess SOD activity included observation by UV-vis spectroscopy of the oxidation of nitroblue tetrazolium (NBT) (68) or the oxidation of a cytochrome c by superoxide (52). Both systems used superoxide from an in situ generator, frequently xanthine oxidase, wherein the complex being analyzed was compared to a calibrated oxidation of the chromophore alone and in the presence of MnSOD. The direct observation of the decrease in the superoxide signal with time by UV-vis is also possible, and superoxide may be introduced as a solution (428) or generated, in some cases, by pulsed radiolysis (79, 80). In these direct observation experiments, the rate of decay of superoxide in the presence of the complex is compared to the rates of decay of superoxide alone and in the presence of one unit of activity of MnSOD. In all cases, the systems are usually referenced, or calibrated, against the same set of conditions with MnSOD. Due to interactions with cytochrome c with components of assay mixtures other than superoxide, false readings of activity were often observed for some early SOD mimics. The NBT, stopped-flow, or pulsed radiolysis techniques have tended to provide the more accurate answers on the ability of reputed MnSOD mimics. To be considered active in any manner with respect to the decay of superoxide in the stopped-flow analyses, Weiss et al. stated that compounds based on their analyses needed to exhibit kcat values in excess of 10B 5 M 1 s 1 (428). 

The autoxidation of hydroxylamine to nitrite also involves a radical chain process (Kono, 1978), and the reaction is carried out at high pH. The assay was originally utilized by Elstner and Heupel (1976) who initiated the autoxidation by O2 generated by the xanthine/xanthine oxidase reaction. Nitrite formation was determined colorimetrically at 530 nm by diazo coupling with a-naphthylamine and superoxide dismutase was found to inhibit end product formation. Kono (1978) developed the assay further by utilizing nitroblue tetrazolium as the... 

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. 

Also in the superoxide anion scavenging assay in the phenazine methosulfate/NADH-nitroblue tetrazolium system, garcinol exhibited potent superoxide anion scavenging activity almost comparable to that of gallic acid and stronger than that of (+)-catcchin [60]. 

The ability of a test compound to induce differentiation of HL-60 cells (derived from a patient with acute promyelocytic leukaemia) is also an important assay of retinoid activity [91-93]. HL-60 cells do not produce superoxide anions upon stimulation by agents like TPA. Differentiated HL-60 cells, however, do produce these anions upon similar stimulation. The presence of these anions can be monitored because of their ability to reduce the yellow nitroblue tetrazolium (NBT) to the water insoluble blue-black formazan. Determination of the concentration (ED50) which results in differentiation of 50% of the cells is the measure of the anti-APL activity of the retinoid. Morphological maturation of the cells can also be checked. 

Recently, the superoxide dismutase activity of low molecular mass copper chelates in the indirect coupled assay systems has been dispute It was demonstrated that copper in CuSO and Cu(II)(gly)2 prevents the ferricytochrome c and nitroblue tetrazolium reduction. This is not virtually new, as it is a well known phenomenon that Cu(II)-salts lead to a reoxidation of ferrocytochrome c and that they are potent inhibitors of xanthine oxidase which is often used as Oj" -generator in indirect SOD assay systems Although the indirect assays may be sometimes inadequate for the measurement of the SOD-activity, there are no doubts that low molecular mass copper chelates have their superoxide dismutase during pulse radiolysis. 

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