Tag Archives: fluorescence

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: danylovych@biochem.kiev.ua

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 Ca2+-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.

Activity of NAD•H-generating enzymes and cytochrome content in mitochondria from rat liver and myocardium under artificial hypobiosis

S. D. Melnychuk, S. V. Khyzhnyak, V. S. Morozova, V. M. Voitsitsky

National University of Life and Environmental Sciences of Ukraine, Kyiv;
e-mail: director@quality.ua

The modification particularities of the structural and functional state of the inner mitochondrial membrane of the rat liver and myocardium were observed in conditions of artificial hypobiosis, which was created using hypoxic and hypercapnic gas medium with a body temperature reduction. Under the artificial hypobiosis the activity of NAD·H-generating enzymes of the Krebs cycle of the liver mitochondria decreases. The established changes of the enzymes activity and cytochromes content of the inner mitochondrial membrane indicate the decrease of the oxidative activity of a respiratory chain, that can be limi­ted on a terminal (cytochrome c oxidase) site and leads to the decrease (by 49% at an average) of the H+-ATPase­ activity of the liver mitochondria. Under the artificial hypobiosis the detected increase of the succinate-KoQ-oxidoreductase activi­ty (by 65% at average) causes the maintaining of the functional activity of a mitochondrial respiratory chain, considering the high (relative to control) cytochrome c oxidase and H+-ATPase activities of the mitochondria of the rats’ myocardium. The structural changes of the inner mitochondrial membrane of the liver and myocardium in experimental conditions are accompanied by the increase of hydrophobicity of tryptophan residues microenvironment and the intramolecular modifications of protein molecules.

Comparison of bioactive aldehydes modifying action on human albumin

I. P. Krysiuk, A. J. Knaub, S. G. Shandrenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
е-mail: iryna-kr@yandex.ua

Protein’s postsynthetic modifications are a cause and a consequence of many diseases. Endogenous aldehydes are one of the main factors of these modifications formation. The human albumin’s modification under some aldehydes influence in in vitro experiment has been investigated. Human albumin (20 mM) was incubated with following aldehydes: ribose, glyoxal, methylglyoxal and formaldehyde (20 mM each) and their combinations in 0.1 M Na-phosphate buffer (pH 7.4) with 0.02% sodium azide at 37 °C in the dark for up to 30 days. We have determined the fluorescent properties of the samples, the content of protein’s carbonyl groups and the redistribution of protein’s molecular weight.
The following ratings of aldehydes from the lowest to the highest effect have been obtained. Fluo­rescent albumin adducts formation: formaldehyde, methylglyoxal, ribose, glyoxal; carbonylation of the protein: ribose, formaldehyde, glyoxal, methyl­glyoxal; polymerization of albumin – the formation of intermolecular crosslinks: ribose, methylglyoxal, glyoxal, formaldehyde. The results indicate that these aldehydes have different capability for protein’s modifications. For example, formaldehyde, having the lowest ability to form fluorescent adducts, shows the highest ability to form protein’s intermolecular crosslinks. Therefore, methods and parame­ters in order to evaluate the protein postsynthetic modification intensity have to be chosen correctly according to carbonyl stress peculiarity in order to evaluate the protein’s postsynthetic modification intensity.

Effect of bioactive aldehydes on gelatin properties

I. P. Krysyuk, N. D. Dzvonkevych, T. T. Volodina, N. N. Popova, S. G. Shandrenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
е-mail: iryna-kr@yandex.ua

Bioactive aldehydes are among main factors of proteins postsynthetic modifications, which are the cause and consequence of many diseases. Comparative study of some aldehydes modifying action on gelatin was carried out in vitro. Gelatin samples (20 mM) were incubated with: ribose, deoxyribose, glyoxal, methylglyoxal, formaldehyde, acrolein (20 mM each) and their combinations in 0.1  M Na-phosphate buffer (pH 7.4) containing 0.02% sodium azide at 37 °C in the dark for 30 days. We investigated the fluorescent properties of these samples and their molecular weight distribution by electrophoresis. It has been revealed that formed adducts had different fluorescence spectra. According to fluorescence intensity these aldehydes were put in order: formaldehyde < methylglyoxal < acrolein < ribose < deoxy­ribose < glyoxal. The electrophoresis results showed fragments of gelatin molecular weight redistribution. By this index, the aldehydes rating was as follows: ribose < deoxyribose < acrolein < glyoxal < formaldehyde < methylglyoxal. Comparison of these two ratings indicates that aldehydes with a lower ability to form fluorescent adducts have higher abili­ty to form intermolecular crosslinks. Therefore, the traditional clinical fluorescent test of a patients’ skin surface for collagen crosslinks determination has to be verified by other tests for proteins postsynthetic modifications.