**4. SDH activity assay**

The succinate is the most efficient energy source, so the SDH activity assay can be an important method for measurement of the yeast vitality in scope to control, e.g. different fermentation processes [19]. SDH activities can be measured *in vitro* in cell lysates or in mitochondrial fraction as well as *in situ* in individual cells. Since SDH is bound to the inner membrane, it is easily isolated along with the mitochondria by different techniques: sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method [20]. The mitochondrial fraction is the source of the enzyme. Since none of the key components can be measured directly, the reaction succinate → fumarate is measured by monitoring the reduction of an artificial electron acceptor. To use an artificial electron acceptor, the normal path of electrons through the mitochondrial electron transport system must be blocked. This is accomplished by adding either sodium azide or potassium cyanide to the reaction mixture. These poisons inhibit the transfer of electrons from cytochrome a3 to the final electron acceptor, oxygen, thus electrons cannot be passed along by the preceding cytochromes and coenzyme Q. Instead, the electrons from SDH-FADH2 can be picked up by an artificial electron acceptor, such as the dye 2,6dichlorophenolindophenol (DCIP). The reduction of DCIP can be followed spectrophotometrically since the oxidized form of the dye is blue and the reduced form is colorless. This reaction can be summarized as

218 Dehydrogenases

[18] (Figure 6).

**4. SDH activity assay** 

UQ pool redox poise under these conditions [13].

Therefore, the SDH, thanks to its unique redox properties, may be a key enzyme to control

Also mutations in genes encoding SDH subunits lead to reduced activity of SDH enzyme. The yeast cells disrupted in *SDH2* (*sdh2*Δ) showed dramatically accumulate succinate resulting in inhibition of at least two α-ketoglutarate dependent enzymes that generate succinate as a by-product. Disruption of complex II activity should alter TCA cycle metabolite levels in the mitochondrial matrix. It was found that neither *sdh1*Δ, nor s*dh2*Δ cells have measurable SDH activity. The succinate accumulates to 8-fold higher levels in *sdh2*Δ cells relative to wild-type cells. Furthermore, complex II + III activity was completely abolished in both SDH mutants without a corresponding compensation in NADH dehydrogenase activity. As a result, complex IV activity was decreased in the SDH mutants

**Figure 6.** Relative concentration of main metabolities of TCA cycle for *sdh1*Δ, nor s*dh2*Δ yeast cells [18].

The succinate is the most efficient energy source, so the SDH activity assay can be an important method for measurement of the yeast vitality in scope to control, e.g. different fermentation processes [19]. SDH activities can be measured *in vitro* in cell lysates or in mitochondrial fraction as well as *in situ* in individual cells. Since SDH is bound to the inner membrane, it is easily isolated along with the mitochondria by different techniques: sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method [20]. The mitochondrial fraction is the source of the enzyme. Since none of the key components can be measured directly, the reaction succinate → fumarate is measured by monitoring the reduction of an artificial electron acceptor. To use an artificial electron acceptor, the normal path of electrons through the mitochondrial electron transport system must be blocked. This is accomplished by adding either sodium azide or potassium

#### SDH-FADH2 + DCIPoxid. → SDH-FAD + DCIPred. + 2H+

The change in absorbance, measured at 600 nm, can be used to follow the reaction over time [21]. To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked. This is accomplished by adding either potassium cyanide or sodium azide to the reaction mixture. The rate of the disappearance of the blue color is proportional to the concentration of enzyme. The change in absorbance of the mixture is measured as a function of time and the enzyme concentration is determined from these data. Enzymatic reactions in yeasts are usually studied in cell-free extracts which requires disruption of cells and as consequence, inactivation of particular enzymes often can be observed. Generally we can conclud that determination of SDH enzyme activity has proved to be a difficult enzyme to extract from respiratory membrane whilst still retaining its *in vivo*  properties. Most of the described extraction procedures were rather drastic and yielded soluble preparations of rather dubious integrity [8].

In recent years quantitative histochemical procedures has been proved to be a powerful research tool, especially in microphotometric assessment *in situ* of the specific activity of dehydrogenases in individual cells. These assays are simple and valid alternative to conventional biochemical techniques. Methods *in situ* can provide the cellular resolution necessary to determine enzyme-specific activities not only in whole cell preparations but also in distinct subcellular compartments [19].

Reduction of various tetrazolium salts by dehydrogenases of metabolically active cells leads to production of highly colored end products – formazans (Figure 7). The history of the tetrazolium salts and formazans goes back 100 years, to when Friese (1875) reacted benzene diazonium nitrate with nitromethane, to produce a cherry-red "Neue Verbindung". This was the first formazan. Nineteen years later, Von Pechmann and Runge (1894) oxidised a formazan to produce the first tetrazolium salt [21].

**Figure 7.** Tetrazolium salt and its coloured formazan.

Many hundreds of tetrazolium salts and formazans were prepared in the following years, but only a handful have found applications in biological research.There is a wide range of tetrazolium salts commonly used in the field of microbiology from the classical ones to the new generation of its derivatives. Among them are: blue tetrazolium chloride (BT), 2,3,5 triphenyl tetrazolium chloride (TTC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), 2,3-bis(2-methoxy-4-nitro-5 sulphophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), 4-[3-(4 idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST1), 2-(piodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) or 2,2'-dibenzothiazolyl-5,5'-[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4' biphenyl) ditetrazolium, disodium salt (WST-5) [19, 22, 23].

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 221

**Figure 9.** Yeast cells after reaction with 2,3,5-triphenyl tetrazolium chloride (TTC). A – without permeabilization; B – with permeabilization by 0.05% digitonin. Images of fluorescence microscopy.

(A) (B)

**Figure 10.** Yeast cell after reaction with 2,3,5-triphenyl tetrazolium chloride (TTC). A – without permeabilization; B – with permeabilization by 0.05% digitonin. Images of scanning microscopy.

(A) (B)

activity and ATP content were observed during aging of tested yeast strains [19, 23].

**5. The role of SDH in human disease** 

with that of ATP content in yeast cells. Significant decreasing of succinate dehydrogenase

*Saccharomyces cerevisiae* is a simple eukaryotic organism, with a complete genome sequence. Many genetic tools that have been created during these years, including the complete

In the case of enzymatic reaction conducted *in situ* the plasma membrane forms a barrier with low degree of penetration. Therefore, cell permeabilization, e.g. by digitonin, is recommended as an alternative method for the study of intracellular enzyme activities. According to the results obtained by Berlowska et al. [23] digitonin was effective in membrane permeabilization without negative influence on cell morphology. After digitonin treatment, the visible formazan crystals were observed inside the yeast cells, but not outside them (Figures 8-10 A, B).

The formazan products are water-insoluble, but readily diffuses out of yeast cells after solubilization in DMSO. Good correlation (R2=0,97) between BTf absorbance intensity after DMSO extraction and number of yeast cells was seen. Linear correlation was observed in the concentration range of yeast cells from 9107 to5108 per sample. For yeast cell concentrations below 1107 per sample the formazan color intensity signals were too low to detect with good precision. The results obtained for SDH activity were in good agreement

**Figure 8.** Yeast cells after reaction with blue tetrazolium chloride (BT). A – without permeabilization; B – with permeabilization by 0.05% digitonin. Images of light microscopy.

**Figure 9.** Yeast cells after reaction with 2,3,5-triphenyl tetrazolium chloride (TTC). A – without permeabilization; B – with permeabilization by 0.05% digitonin. Images of fluorescence microscopy.

**Figure 10.** Yeast cell after reaction with 2,3,5-triphenyl tetrazolium chloride (TTC). A – without permeabilization; B – with permeabilization by 0.05% digitonin. Images of scanning microscopy.

with that of ATP content in yeast cells. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of tested yeast strains [19, 23].
