Cytochrome c assay

Phenoxy radical species were believed to be present on the fibre surface, and superoxide was also identified in a cytochrome c assay. However, the presence of superoxide... 

The complex of manganese with desferrioxamine B (429-432), reported to be a MnSOD mimic in 1987, illustrates this point. In the initial studies, the cytochrome c assay was applied, and this compound was reported to be a catalytic MnSOD mimic. Follow-up studies by Riley and co-workers by stopped-flow techniques showed that this complex interacts at best on a stoichiometric level with superoxide, but it could not be considered to be a catalyst and was termed inactive (428). This occurred due to interactions with cytochrome c wherein the manganese complex interfered with the reduction of cytochrome c, leading in effect to a false positive. Thus, although some... 

Numerous indirect assays, such as the cytochrome c assay, have been used in attempts to measure the SOD activity of putative SOD mimics,However, these assays, which typically rely on a spectrophotometric change of a redox indicator to measure superoxide levels, cannot kinetically distinguish between a catalytic dismutation of superoxide and a stoichiometric interaction of superoxide with the putative SOD mimic. Moreover, the indirect assays are prone to false positives or false negatives respectively, when the putative SOD mimic oxidizes or reduces the spectrophotometric indicator. 

Fig. 2. Heat denaturation of purified bovine erythrocyte Cu2Zn2Superoxide dismutase. (O) SOD prepared by treatment with chloroform/ethanol, (A) aqueously isolated enzyme and ( ) the same enzyme stored for three months as lyophilized powder at room temperature. When freshly prepared, all fractions of aqueously isolated superoxide dismutase showed the same behaviour as (A) regardless of the age of the animal and the isoelectric point. SOD-activity was estimated using the cytochrome c assay...

Pandiaraj, M., Alby, RJ3., Madasamy, T., Vairamani, K., Arya, A., Sethy, N.K., Kalpana, B., Karunakaran, C., 2014. A cost-effective volume miniaturized and microcontroller based cytochrome c assay. Sens. Acmators A 220, 290—297. 

Figure 7 Differentiation of HL-60 cells by the incubation with dimethylsulfoxide [DMSO] (a), and the assay of superoxide release in the DMSO-differentiated HL-60 cells by cytochrome C method (b).

Assayed by the luminol chemiluminescence method (Puget and Michelson, 1974). One unit corresponds to 0.35 unit of the cytochrome c method (McCord and Fridvich, 1969). 

In the presence of cytochrome C, changes in transmittance at 550 nm reflect oxidant production as superoxide reduces the cytochrome C. This is usually done as an absorbance assay in a spectrophotometer, but it can be performed as a transmittance assay on the SLM fluorometer (20). 

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] ... 

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 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. 

It has earlier been suggested to make cytochrome c a more specific reagent for superoxide detection by its acetylation or succinoylation [9-11], It was proposed that acetylation and succinoylation must cause a greater decrease in the reaction of cytochrome c with NADPH cytochrome P-450 reductase than with superoxide due to a decrease in the electrostatic charge of native cytochrome c [12]. However, the rate constant for the most selective succinoylated cytochrome c became about 10% of native cytochrome [13], making this assay even less sensitive. 

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.

Hence, 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. 

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. 

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