Superoxide dismutase assay

Isolation, Purification, Characterization, and Assay of Antioxy-genic Enzymes Isolation and characterization of superoxide dis-mutase, 105, 88 superoxide dismutase assays, 105, 93 assays of glutathione peroxidase, 105, 114 catalase in vitro, 105, 121 assays of lipoxygenase, 105, 126. 

McCord, J. M., Crapo, J. D., Fridovich, I. Superoxide dismutase assays A review of methodology. In Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., Fridovich, I., eds.), London-New York-San Francisco, Academic Press, 1977, pp. 11-17 Ysebaeit-Vanneste, M., Vanneste, W. H. Analyt. Biochem. 107, 86 (1980)... 

Flohe, L. and Otting, F. (1984) Superoxide dismutase assays, Methods Enzymol. 105, 93-105. 

Oka, S., Ogino, K., Matsuura, S., Yoshimura, S., Yamamoto, K., Okazaki, Y., Takemoto, T., Kato, N., and Uda, T Human serum immuno-reactive copper, zinc-superoxide dismutase assayed with an enzyme monoclonal immunosorbent in patients with digestive cancer. Clin. Chim. Acta 182, 209-220 (1989). 

Or From Tetrabutyl-Ammonium Superoxide. Tetrabutyl-ammonium superoxide was found convenient for the superoxide dismutase assay since it dissolved readily without decomposition in N,N-dimethyl form-amide (70). Infusions of this solution into a cuvette containing aqueous oxidized cytochrome c reduced the available cytochrome c. In the presence of different erythrocuprein concentrations the reduction rate was progressively diminished. According to McCord and Fridovich (70) an enzyme unit was defined as 50% inhibition of the rate of reduction of cytochrome c. Alternatively tetranitromethane was found appropriate for monitoring Or (70, 136—148) where the stable nitroform anion C(N02)3 is being formed (Equ. (a)) ... 

Proof of the generation of Or during electrolysis of aqueous solutions was obtained in the laboratory of Fridovich (206). The OI catalysed oxidation of adrenaline served as a monitor which could be inhibited by superoxide dismutase. Ultrasonication of buffered aerated solutions gave rise to the formation of Or which could be detected using the cytochrome-c reductase assay (207). This sonication induced cyto-chrome-c reduction was also inhibited by native erythrocuprein. Another sensitive superoxide dismutase assay using the reduction of nitro blue tetrazolium by Or was developed by Beauchamp and Fridovich (208). This assay allowed the detection of erythrocuprein in the ng/ml region. During the metalloenzyme conference in Oxford, 1972, Fridovich summarized the basic facts on superoxide dismutase (erythrocuprein) (209). 

The reactivity of native erythrocuprein was higher compared to the superoxide dismutase activity shown in Table 12. Of utmost importance was the observation that the model chelates and CUSO4 were virtually inactive compared to the native enzyme. The difference was 4 orders of magnitude which implies a much higher specificity for this enzymic reaction of the cupreins. The powerful reactivity of erythrocuprein is further demonstrated by the fact that the apoprotein displayed a detectable enzymic activity due to traces of copper which were undetectable by atomic absorption measurements or EPR spectroscopy. No such difference between apoprotein and the boiled native enzyme was observed using the superoxide dismutase assay. 

The oxidation of catecholamines like epinephrine has been widely used as source for superoxide dismutase assays. Upon oxidation the catecholamines are transformed to the coloured product adrenochrome. The rate of oxidation by superoxide is inhibited in the presence of superoxide dismutases Likewise the autoxidation of catecholamines at alkaline pH-values is diminished Intriguingly, low molecular mass copper complexes which display superoxide dismutase activity accelerate the autoxidation Therefore, the interaction between superoxide and catecholamines and its inhibition by SOD is thought not to be a simple chemical reactionRecently, this reaction was investigated in more detail Whilst adrenalin autoxidation is very specifically inhibited by SOD, the reaction with other catecholamines like noradrenalin or dihydroxyphenylalanine, having no free amino group, is much less specific. Only 20 % inhibition by CujZnjSuperoxide dismutase are observed. The autoxidation reaction itself is very complex (Scheme 2) and still not fully understood. 

Another advantage of pulse radiolysis lies in the evaluation of the catalytic scavenging of superoxide by low molecular mass complexes. Unlike the polarographic method, another direct superoxide dismutase assay, the reaction parameters of such compounds can be easily obtained. 

Nakano, M. (1990). Assay for superoxide dismutase based on chemiluminescence of luciferin analog. Method. Enzymol. 186 227-232. 

Oxidant production is measured with the fluorogenic substrate para-hydroxyphenylacetic acid (PHPA) in the presence of superoxide dismutase and peroxidase (9). Under these conditions, superoxide is converted to H2O2 by the superoxide dismutase, and two molecules of PHPA are converted to a fluorescent diadduct by H2O2 and peroxidase. Similar assays have been devised using homovanillic acid (16) or scopoletin (17) instead of PHPA. 

Misra, H.P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry 247 3170-3175. 

C. Beauchamp and I. Fridovich, Superoxide dismutase. Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287 (1971). 

Popov IN, Lewin G and von Baehr R. 1987. Photochemiluminescent detection of antiradical activity. I. Assay of superoxide dismutase. Biomed Biochim Acta 46(11) 775—779. 

The basis of this assay was first used to measure the activity of superoxide dismutase (SOD) using a xanthine/xanthine oxidase 02"-generating system. O2 generated via this enzyme will reduce feni (oxidised)-cytochrome c, but SOD (which has a much higher affinity for O2" than cytochrome c) will prevent this reduction. Babior, Kipnes and Cumutte (1973) modified this technique to provide a specific assay to measure O2 production by activated neutrophils. Thus, 02" reduces cytochrome c (measured by an absorbance increase at 550 nm), but this reduction will be blocked by the addition of exogenous SOD (Fig. 5.10). 

Figure 5.10. Cytochrome c reduction by 02 Production of 02 from activated neutrophils may be assayed using cytochrome c. Oxidised (Fe3+) cytochrome c can be reduced by 02" to form Fe2+-cytochrome c, which absorbs at 550 nm thus, in a mixture of activated neutrophils and cytochrome, absorption increases at 550 nm are due to 02 production. Superoxide dismutase (SOD) has a higher affinity for 02 than does cytochrome c thus, the addition of SOD to activated neutrophil suspensions will prevent the reduction of cytochrome c. SOD-inhibitable cytochrome c reduction is therefore a direct measure of the rate of 02 formation.

Minami M, Yoshikawa H (1979) A simplified assay method of superoxide dismutase activity for clinical use. Clin Chim Acta 92 337-342... 

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... 

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