Superoxide dismutases measurement

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. 

F. Lisdat, B. Ge, E. Ehrentreich-Forster, R. Reszka, and F.W. Scheller, Superoxide dismutase activity measurement using cytochrome c-modified electrode. Anal. Chem. 71,1359—1365 (1999). 

S. Descroix and F. Bedioui, Evaluation of the selectivity of overoxidized polypyrrole/superoxide dismutase based microsensor for the electrochemical measurement of superoxide anion in solution. 

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 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.

OXYGEN, OXIDES 0X0 ANIONS PHOTOOXYGENATION SUPEROXIDE DETECTION/MEASUREMENT SUPEROXIDE DISMUTASES SUPERACID... 

Destruction of nitric oxide by superoxide in the buffers is more likely to account for the short half-life of nitric oxide in vitro. Superoxide dismutase (15-100 U/ml) substantially increased the apparent half-life of EDRF, strongly suggesting that superoxide contributes to the short biological half-life of nitric oxide. In the perfusion cascade bioassay system, the buffers are bubbled with 95% oxygen, contain 11 mM glucose as well as trace iron plus copper contamination and are incubated under the weak ultraviolet (UV) radiation of fluorescent lights. These are prime conditions for the autoxidation of glucose to form small amounts of superoxide in sufficient amounts to account for the short half-life of nitric oxide in nanomolar concentrations. The rate of reaction between superoxide and nitric oxide is 6.7 X 10 M sec L The shortest half-life of nitric oxide measured is approximately 6 sec. To achieve a half-life of 6 sec, the steady state concentration of superoxide would only need to be 17 pM, calculated as ln(2)/ (6 sec X 6.7 X 10 M" sec )-... 

We have used the nitration of a tyrosine analog with superoxide dismutase to measure the peroxynitrite production from activated rat alveolar macrophages (Fig. 38). The estimated rate of peroxynitrite synthesis was estimated to be 0.1 nmol/10 cells/min (Ischiropoulos et al., 1992a). The rate of nitration was the same whether native Cu,Zn-superoxide dismutase or the phenylglyoxyl-H202 modified superoxide dismutase (which is >99% inhibited with respect to its superoxide scavenging activity) was used (Fig. 39). Three other independent but indirect estimates of peroxynitrite formation were consistent with the superoxide... 

Plasma malondialdehyde-like material, an indicator of lipid peroxidation, is increased in conditions of ischaemia, such as stroke [83, 84] and myocardial infarction [85]. Mitochondria extracted from hearts of vitamin-E-deficient rabbits showed a decreased mitochondrial function and an increased formation of oxygen radicals associated with a reduced superoxide dismutase activity. This was partially reversed by addition of vitamin E in vitro [86]. Measurement of in vitro susceptibility to lipid peroxidation in cardiac muscle from vitamin-E-deficient mice showed a highly significant negative correlation between the concentration of vitamin E and in vitro lipid peroxidation. The results indicate that short-term vitamin E deficiency may expose cardiac muscle to peroxidation injuries [ 87 ]. In rats, treatment for 2 days with isoprenaline increased lipid peroxide activity, as measured by malondialdehyde levels, in the myocardium. Vitamin-E-deficient animals were even more sensitive to this effect, and pretreatment with a-tocopheryl acetate for 2 weeks prevented the effect induced by isoprenaline. The authors [88] propose that free-radical-mediated increases in lipid peroxide activity may have a role in catecholamine-induced heart disease. 

The structure and enzyme kinetics of bovine erythrocyte superoxide dismutase are reviewed. The protein has a novel imidazolate-bridged copper(II)-zinc(II) catalytic center in each of two identical subunits. Since a C /Cu1 redox couple is responsible for the dismutase activity of the enzyme, the role of zinc is of interest. Both 220-MHz NMR measurements of the exchangeable histidine protons and chemical modifications using diethylpyrocarbonate demonstrate that zinc alone can fold the protein chain in the region of the active site into a conformation resembling that of the native enzyme. Other possible roles for zinc are discussed. Synthetic, magnetic, and structural studies of soluble, imidazolate-bridged copper complexes of relevance to the 4 Cu(II) form of the enzyme have been made. 

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