Superoxide dismutase activity detection

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. 

Soluble proteins containing b-type hemes have not been detected in anaerobes, with the exception of catalase all other examples of b-type cytochromes known so far are membrane-associated. A catalase was isolated from D. vulgaris [87], and its activity has also been detected in other strains of Desulfovibrio species. Superoxide dismutase activities have also been detected in these bacteria [87]. As mentioned in the introduction, the finding of catalase and superoxide dismutase in an anaerobe is puzzling. It is not known whether these enzymes are just used for detoxification or indicate the presence of adaptation capabilities to oxygenic environments. 

Figure 3.11. Detection by polyacrylamide gel-electrophoresis of superoxide dismutase (A) and peroxidase (B) activities in crude extracts of P. coccoides (1), P. shermanii (2) and P. globosum (3). A gel stained for superoxide dismutase activity according to Beauchamp and Fridovich (1971), Stain solutions contained no KCN. B gel stained for peroxidase activity according to Gregory and Fridovich (1974). From Kraeva and Vorobjeva (1981b).

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 process of the superoxide-dependent PCL that can be inhibited by enzyme superoxide dismutase (SOD) is shown in Figure 2. Luminol can be replaced by lucigenin. In this case, only the first maximum is detected. This variant of the system is useful for SOD activity measurements. The system is very sensitive and rugged therefore, it is even possible to perform the enzyme determination in whole blood [22],... 

The bacterium Lactobacillus plantarum and its closest allies are unusual in that they are aerobic organisms but do not produce a superoxide dismutase. This bacterium instead accumulates Mn(II) to an intramolecular level on the order of 25 mM (150-152). In vitro studies indicated that Mn(II) formed a complex with lactate which possessed significant superoxide activity (153). These bacteria are additionally unable to produce heme and, consequently, when grown in the absence of heme, produce a hemeless catalase, or pseudocatalase (154-158). Unlike heme-containing catalases, the enzyme is not inhibited by cyanide or azide, and the addition of either Mn or Fe into the growth medium increased the amount of the pseudocatalase present. However, neither of the metals could be detected in partially purified enzyme assays (157). 

There is considerable body of (indirect) evidence which makes oxidative stress one of the best accepted hypothesis for explaining the cause of Parkinson s disease. For example, the Fe(II)/Fe(III) ratio in the substantia nigra is shifted from 2 1 in the normal brain to 1 2 in Parkinsonian brain.131,132 In the Parkinsonian brain several enzymes which constitute the antioxidative defence mechanisms (glutathione peroxidase, catalase) have a decreased activity, while the activity of superoxide dismutase is increased, relative to the normal brain.133 Furthermore, specific products of radical damage, such as lipid hydroperoxides, were detected at a 10-fold increased level in the Parkinsonian brain.134... 

These side reactions of superoxide dismutase are generally of no biological significance (except for a possibility in pathology, see Sect. 1.9). Nevertheless, they may be important under peculiar conditions e.g., due to its peroxidase activity, SOD may increase rather than decrease oxidation of a detector of reactive oxygen species [68]. The oxidation rate of a popular probe used for detection of reactive oxygen species, 2, 7 -dichlorofluorescin, is considerably augmented by CuZnSOD, especially in the presence of bicarbonate [67]. 

The indirect methods of assaying for superoxide dismutase are the most widely used routine assays because the reagents used are inexpensive, widely available, and usually very sensitive to detection. However, these types of assay are quite susceptible to interferences. Some of these interferences are unfortunately ignored, with the consequence that various discrepancies arise in the results obtained from the various laboratories. These assays can operate at a very low steady-state level of O2 and are considered to be indirect negative assays when superoxide dismutase inhibits the formation of products and indirect positive assays when the enzyme accelerates the formation of products. In the indirect assays a unit of enzyme activity is generally defined as the amount of the enzyme that inhibits the reaction by 50%. 

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. 

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