ISSN 2409-4943. Ukr. Biochem. J., 2016, Vol. 88, N 1 UDC 576.311.347:577.113.3 doi: http://dx.doi.org/10.15407/ubj88.01.031 EVALUATION OF FUNCTIONING OF MITOCHONDRIAL ELECTRON TRANSPORT CHAIN WITH NADH AND FAD AUTOFLUORESCENCE H. V. DANYLOVYCH Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv; e-mail: [email protected] We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to 2+Ca - dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain. K e y w o r d s: mitochondrial electron transport chain, NADH, FAD, fluorescence, inhibitors, myometrium of rats. itochondria are the key players of cell transport chain (ETC) (Fig. 1). NADH and FADH2 ener gy metabolism, programmed cell oxidation leads to translocation of protons through M death and oxidative stress. Polarogra- complexes I, III and IV of the mitochondrial inner phy with Clark electrode is a widely used method membrane into intermembrane space. Proton gradi- for evaluation of functional activity of mitochondria ent and ADP control rate of ATP synthesis by mi- through measurement of oxygen consumption rate tochondria. Thus, changes in redox state of NADH by suspension of the organelles of permeabilized and FADH 2 are indicators of mitochondria energet- cells in various functional states [1-5]. This method ics [6-8]. requires specific equipment and large quantities of NADH is essential as a coenzyme in catalytic experimental material, yet it does not provide in- reactions of the primary metabolic pathways and formation on the biochemical processes underly- also is a dominant component of cellular autofluo- ing changes in mitochondrial oxygen consumption. rescence. After it has donated electrons, primary to Therefore, research and development of simple, re- the mitochondrial ETC, NAD+ does not fluoresce. liable and informative biochemical approaches to Unlike NADH, FADH2 has no intrinsic fluorescence evaluation of functional activity of particular com- and FAD has it, which allows for visualization of re- plexes of mitochondrial electron-transport chain are dox state of these nucleotides using optical methods needed. without employing fluorescent probes. Therefore, NADH (nicotinamide adenine dinucleotide) changes in fluorescence of pyridine and flavin nu- and FADH 2 (flavin adenine dinucleotide) are the pri- cleotides may be used to evaluate mitochondria ener- mary electron carriers within mitochondrial electron gy-generating efficiency [8-10]. 31 ISSN 2409-4943. Ukr. Biochem. J., 2016, Vol. 88, N 1 Evaluation of redox state of pyridine nucleo- Thus, the aim of the present study was to inves- tides is widely used in biochemical research, in par- tigate NADH and FAD fluorescence as biomarker for ticular in studies on consequences of oxidative stress evaluation of functioning of isolated mitochondria [11-13], cell death detection [8], estimation of level of (in the case of myometrium of rats) under the effect postsynaptic neuronal activation [10, 14]. The corre- of modifiers of ETC and ion transport. sponding experiments were made on carcinoma cells [15], lung cells [16], heart tissue [17-19]. Changes in Materials and Methods redox state of pyridine nucleotides as the primary Mitochondria isolation from myometrium. Mi- cause of disruptions in mitochondrial respiration tochondria samples were isolated from myometrium under fluoroacetate toxicity have been studied on of non-pregnant rats by differentiation centrifuga- ascitic Ehrlich carcinoma and rat liver mitochondria tion, as described [24]. Rats were anesthetized by [20]. diethyl ether inhalation and decapitated. The animal NADH has been described to fluoresce at 450- experiments were conducted in accordance with 460 nm on excitation at 340-350 nm. FAD emits at guidelines of the European Convention for the Pro- 530-540 nm on excitation at 450 nm [21, 22]. The tection of Vertebrate Animals used for Experimental amount of data on changes in fluorescent signal of and Other Scientific Purposes (Strasbourg, 1986). NADH under influence of ETC effectors in isolated For the duration of the experiment the isolated mitochondria [5, 12, 20, 23] is limited, and there is mitochondria fraction was kept on ice. Protein con- no data on changes in FAD signal. There is also no tent in mitochondria fraction was determined by the information on the corresponding studies on smooth standard procedure after Bradford [25]. The mean muscle cell and mitochondria. content was 2 mg/ml. Fig. 1. Electron transfer and inhibitors of respiratory complexes in electron transport chain of mitochondria. Complex I – NADH-ubiquinone oxidoreductase, II – succinate dehydrogenase, III – ubiquinone-cytochrome-c oxidoreductase, IV – cytochrome c oxidase, Q – ubiquinone, QH2 – ubiquinol. Original diagram 32 H. V. Danylovych Detection of NADH and FAD fluorescence in mined two maximums of excitation – at 280 nm and mitochondria using spectrofluorometry. The rela- 350 nm. As shown by others, the excitation maxi- tive values of NADH and FAD intrinsic fluorescence mum at 280 nm is due to tryptophan residues in pro- were determined with Quanta Master PTI (Canada) tein. At 280 nm excitation tryptophan fluoresces at using FelixGX 4.1.0.3096 software. The detection 300 nm, yet chemical microenvironment shifts this was done in 2 ml of the following medium: 20 mM to a significantly longer wavelength [21, 22, 27, 28]. HEPES (pH 7.4 at 37 °C), 2 mM K+-phosphate buffer In this case the maximum of tryptophan fluores- (pH 7.4 at 37 °C), 120 mM KCl, 5 mM pyruvate, cence may be at 400 nm (Fig. 2). 5 mM succinate. The aliquots of mitochondria con- The maximums of excitation and emission for tained 100 μg of protein. NADH in mitochondrial fraction from rat uterus The relative fluorescence of NADH and FAD myocytes are 350 nm and 450 nm, correspondingly (Fig. 2, B, C). The maximums of excitation and fluo- was calculated as (F–F0)/F0, where F0 is the initial fluorescent signal and F is the signal at the corre- rescence of FAD were at 450 nm and 533 nm (Fig. 3, sponding time period. B, C), which is in accordance with data by others In experiments on rotenone, antimycin and [8-10, 16, 21, 22]. Moreover, excitation and emission A23187, a Ca2+-ionophore, the intrinsic fluorescence spectra of the investigated adenine nucleotides are of these compounds was taken into account. matching those of working solutions of the corre- Estimation of hydrodynamic diameter of mito- sponding chemically pure nucleotides (Fig. 2, A; 3, chondria. Hydrodynamic diameter of mitochondria A). It must be noted that NADH binding to proteins was assayed with ZetaSizer 3 multi-angle particle prevents contact (stacking) between adenine and re- size analyzer (Malvern Instruments, Great Britain) duced nicotinamide group, which causes increase in equipped with computing correlator type 7032 and quantum output of fluorescence, although the corre- sponding effect for FAD has not been detected [21]. He-Ne laser LHN-111 with λ = 633 nm and 25 mW Changes in NADH and FAD fluorescence in output. Autocorrelation of laser light scattering on mitochondrial fraction in medium with substrates mitochondria suspension was registered for 1 min, of respiration. We introduced 5 mM pyruvate, a in 10 series under 90° scattering angle. The autocor- substrate of pyruvate dehydrogenase complex that relation function was calculated with standard PCS- produced NADH for ETC, and 5 mM succinate, a Size mode v. 1.61 software. The incubation medium substrate of FAD-dependent succinate dehydroge- (1 ml) was of the composition described above. Mi- nase, into incubation medium in order to produce tochondrial function was aliquoted at 50 μl, which the energized state of mitochondria. corresponded to 50 μg of protein. NADH fluorescence decreased in time in the We used the following reagents: HEPES, presence of respiratory substrates, which indicates sodium pyruvate, sodium succinate, rotenone, anti- increase in NAD+ content resulting from functio ning mycin A, CCCP, NaN , A23187, ATP, bovine serum 3 of NADH-ubiquinone oxidoreductase (complex I) albumin (Sigma, USA), mineral salts of local origin. (Fig. 4, A). FADH content also decreased, with cor- The reagents were dissolved in ddH O with specific 2 2 responding increase in FAD fluorescence, which re- conductance no higher than 1.5 μS/cm, as measured flects the activity of succinate dehydrogenase com- by OK-102/1 conductometer (Hungary). plex (complex II) of ETC (Fig. 4, B, C). Thus, we Data are presented as mean ± SEM; significant observed reciprocity in changes of fluorescence of differences between group were evaluated by using flavin and pyridine adenine-nucleotides under nor- Student’s t-test with P < 0.05 [26].