Cytochrome c reduction assay

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). 

SOD-inhibitable reduction of cytochrome c is thus now widely used to measure 02 production by activated neutrophils. It is an extremely-selective method, being specific for O2 , and is easily measured spectrophotometrical-ly. Because SOD can convert two molecules of O2 into one molecule of H2O2 and one molecule of O2 (reaction 5.1), catalase (which degrades H2O2 

this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

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Cytochrome c Reduction Assay Using a HL-60 Ceii Cuiture System... 

It is extremely important that the interaction of quinones with XO (Reaction (3)) is reversible that can lead to receiving erroneous results at the measurement of superoxide production by SOD-inhibitable cytochrome c reduction [28,29] (see also Chapter 27). Lusthof et al. [30] demonstrated that 2,5-bis(l-aziridinyl)-l,4-benzoquinones are directly reduced by XO. Interestingly at quinone concentrations greater than 25pmol I 1, quinones entirely suppressed one-electron reduction of dioxygen, and cytochrome c was completely reduced by the semiquinones formed. It is well known that cytochrome c and lucigenin are effective superoxide scavengers and due to that, these compounds are widely used in the quantitative assays of superoxide detection. Nonetheless, under certain experimental conditions they can be directly reduced by XO [31]. 

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] ... 

The efficiency of superoxide assays strongly depend on the nature of superoxide producers. Significant difficulties arise in the detection of superoxide in cells and tissue. Cytochrome c is unable to penetrate cell membranes and therefore, can be used only for the measurement of extracellular superoxide. Furthermore, SOD-inhibitable cytochrome c reduction is difficult to apply in nonphagocytic cells and tissue due to the complications of measuring low rates of superoxide release, direct reduction of cytochrome c by cellular enzymes, the reoxidation of reduced cytochrome by hydrogen peroxide, etc. [8], Moreover, in nonphagocytic cells superoxide is formed exclusively inside the cells and is not released outside as in phagocytes. These circumstances severely limit the number of analytical methods, which can be used for superoxide detection in vasculature. 

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.

Because electrode measurements of O2 uptake can detect intra- and extracellular oxidase activity, this assay can be used to measure the respiratory burst elicited by soluble and particulate stimuli. What is somewhat surprising is that, during stimulation of neutrophils with agonists such as fMet-Leu-Phe, the activated O2 uptake profile is biphasic (Fig. 5.11c). A rapid burst of O2 uptake (which coincides with measurements of cytochrome c reduction) is followed by a more sustained activity of lower magnitude. 

Complex in (ubiquinohcytochrome c oxidoreductase) reduces cytochrome c and oxidizes reduced CoQ. In addition to these substrates, the assay contains a strong complex IV inhibitor (e.g. potassium cyanide) to prevent re-oxidation of reduced cytochrome c. The complex III activity can be derived from the rate of cytochrome c reduction, which can be followed at 550 nm. The specificity of the assay is determined by measuring in the absence or presence of antimycin A, a specific inhibitor of complex III activity. 

Bagchi et al. (1997) studied the oxygen free-radical scavenger ability of grape seed proanthocyanidins using a chemiluminescence assay and cytochrome c reduction. The results revealed that on a weight basis (100 mg/L), the grape seed proantho-cyanidin extract was a better inhibitor of both superoxide anion and hydroxyl radical than vitamin C and vitamin E succinate. 

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). 

However, to be a quantitative assay of superoxide detection, Reaction (1) had to be an exothermic reaction, i.e., the difference between the one-electron reduction potentials of reagents AE° = / °[02 /02] / °[A /A] must be <0. In this case the rate constants of Reaction (1) will be sufficiently high (10s—109 1 mol 1 s ). Among traditionally applied assays, three compounds satisfy this condition cytochrome c, lucigenin, and tetranitromethane (Table 32.1). 

The original demonstration of APS reductase in T. thioparus used the methylviologen-dependent assay of APS cleavage to AMP and sulfite (Peck 1960), but in the oxidation of thiosulfate the reaction proceeds in the oxidative direction (Eq. 15.4), forming APS. This is the thermodynamically favorable direction of the reaction. Later work showed that APS formation by the reductase could be coupled to the reduction of ferricyanide or to cytochrome c (Peck et al. 1965 Lyric and Suzuki 1970), thereby showing the thermodynamic feasibility of APS as an intermediate in the oxidation pathways for sulhte and thiosulfate. 

Shown in Figure 6 is in vitro cytotoxic activity of PIPAAm-PBMA micelles loaded with ADR or micelles without ADR at 29°C (below the LCST) and at 37°C (above the LCST) compared with that of free ADR. In vitro cytotoxic activity was measured using bovine aorta endothelial cells. Bovine aortic endothelial cells were obtained as previously reported using dispase for cell dissociation from freshly harvested bovine aorta [13]. The cells plated at a density of 3 x cells/well, were exposed with free ADR or micelles loaded with ADR at below and above the LCST for 5 days. In order to assay cytotoxicity of the free ADR or micelles loaded with ADR, culture medium was replaced with 10% FBS-supplemented phenol red-free DMEM containing 10% alamar Blue, a dye that is subject to reduction by cytochrome c activity and changes the color from blue to red [38]. After 4-hour incubation, reduction of the dye was estimated by absorbance at 560 and 600 nm. PIPAAm-PBMA polymeric micelles loaded with ADR showed higher cytotoxic activity than that of free ADR at 37°C (above the LCST)... 

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