In this paper, using fluorescence and confocal microscopy, we study the content of the flavin adenine nucleotide (FAD) cofactor and enzymatic NAD(P)H-oxidase complexes (with fluorophores Annexin V‑ FITC, 7-AAD (7-aminoactinomycin D), EtBr) under conditions of apoptosis caused by sodium anphen with hydrogen peroxide in healthy mouse splenocytes and Lewis carcinoma tumor cells. The use of fluorescence microscopy allows observing and quantifying the apoptotic effect of sodium anphen and hydrogen peroxide, and it also allows visualizing the metabolic changes in the cell, including the increased fluorescence of FAD in tumor cells and NAD(P)H-oxidase complexes in splenocytes. The data obtained indicate the possibility of using sodium anphen in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells.

It is known that the presence of redox-inactive metals in the active center of an enzyme has a significant effect on its activity. In this regard and for other reasons, the effect of redox-inactive metals on redox processes, such as electron transfer, oxygen and hydrogen atom transfer, as well as the breaking and formation of O–O bonds in reactions catalyzed by transition metals, has been widely studied. Many questions about the role of redox-inactive metals in the mechanisms of these reactions remain open. In this paper, the mechanism of catalysis by bi- and triple hetero-binuclear heteroligand complexes including Ni and redox-inactive alkali metals ((A) {Ni(acac)2∙L2} and (B) {Ni(acac)2∙L2∙PhOH} (L2 = MSt (M = Li, Na, or K)) in the process of the selective oxidation of ethylbenzene by molecular oxygen into α-phenyl ethyl hydroperoxide is considered. The activity of A and B complexes towards O2, ROOH, and RO2• radicals was studied. Based on kinetic data, we suggest that the high catalytic efficiency of B triple complexes in oxidation processes may be associated with the role of outer-sphere regulatory interactions, with the formation of stable supramolecular structures due to intermolecular H bonds. This assumption was confirmed using the AFM method. Prospects for studying catalysis by complexes ({Ni(acac)2∙L2} and {Ni(acac)2∙L2∙PhOH}) that are models of NiARD (Ni-Acyreductone dioxygenase) are discussed.

For the first time AFM (atomic-force microscopy) was used to record significant changes in the geometric parameters of the image of erythrocytes in vitro under conditions of glycolytic starvation (ATP (Adenosine triphosphate) deficiency). The difference in the action of antioxidants, phenosan K, and Ihfan-10 on erythrocytes that we detected with AFM seems to be mainly due to their difference in hydrophobicity. We used the AFM method to research the self-organization of the components of the active center of P450 (Porphyrin-450) metalloenzymes that are part of a class of hemoproteins with functions of affinity to molecular oxygen O2. Stable supramolecular nanostructures in the form of triangular prisms based on the iron porphyrin complex with amino acids due to self-assembly involving intermolecular hydrogen bonds were received. A possible scheme for the formation of such structures is proposed.

The article analyzes the role of hydrogen bonds and supramolecular structures in enzyme catalysis and model systems. Hydrogen bonds play a crucial role in many enzymatic reactions. However, scientists have only recently attempted to harness the power of hydrogen bonds in homogeneous catalytic systems. One of the newest directions is associated with attempts to control the properties of catalysts by influencing the “second coordination sphere” of metal complexes. The role H-bonding, and the building of stable supramolecular nanostructures due to intermolecular H-bonds, based on catalytic active heteroligand iron (Fe) or nickel (Ni) complexes formed during hydrocarbon oxidations were assessed via the AFM (Atomic-force microscopy) method, which was proposed and applied by authors of this manuscript. Th is article also discusses the roles of hydrogen bonds and supramolecular structures in oxidation reactions catalyzed by heteroligand Ni and Fe complexes, which are not only effective homogeneous catalysts but also structural and functional models of Oxygenases.

The role of ligands in the regulation of the catalytic activity of Ni-complexes (Ni(acac)2) in green process-selective ethylbenzene oxidation with O2 into α-phenyl ethyl hydroperoxide is considered in this article. The dual function of phenol (PhOH) included in the coordination sphere of the nickel complex as an antioxidant or catalyst depends on the ligand environment of the metal. The role of intermolecular H-bonds and supramolecular structures (AFM method) in the mechanisms of selective catalysis by nickel complexes in chemical and biological oxidation reactions is analyzed.

The possibility of using the NO sodium μ2-dithiosulfato-tetranitrosyldiferrate tetrahydrate (TNIC-thio) as a drug preventing mitochondrial dysfunction under stress was studied. The properties of TNIC-thio were studied on the mitochondria of 5-day-old etiolated pea seedlings. The functional state of mitochondria was determined under conditions of heat shock (HS) and treatment of seedlings with TNIC-thio. Heat shock activated lipid peroxidation and led to swelling of mitochondria and changes in the content of C18 fatty acids (FAs): the C18 FA unsaturation index in mitochondrial membranes decreased from 1.42 ± 0.01 to 1.22 ± 0.01. Changes also took place in the C20 FA content: the 20:3ω6 content decreased by almost 43.6%, and the 20:0 content decreased by 1.3 times. The change in the pool of unsaturated FAs affected the bioenergetic characteristics of mitochondria: an almost 1.5-fold drop in the rates of oxidation of NAD-dependent oxidation substrates was shown. Treatment of pea seeds with 10 –6 M TNIC-thio was accompanied by prevention of lipid peroxidation (LPO), prevention of changes in the composition of FA membranes and mitochondrial morphology, and restoration of the bioenergetic characteristics of these organelles. The drug could help maintain the functional state of mitochondria under stress by preventing LPO. It perhaps resulted in the preservation of the efficiency of energy processes in the cell, which probably increased the organism’s resistance to changing environmental conditions.

The apoptotic effect of the antioxidant anphen sodium, a spatially hindered phenol, which has antitumor activity—in particular, inhibiting the development of tumor cells in sarcoma—has been studied. It was found that the administration of anphen sodium (10 –4 M) into a cell culture of Lewis carcinoma of mice already after 1–1.5 h led to the exposure of phosphatidylserine and the beginning of the process of apoptosis in the cells (according to the fluorescence of annexin V-FITC). With the combined action of H 2 O 2 (5 μM) and anphen sodium, the permeability of cells to acridine orange increased, and the number of apoptotic cells also increased to 80–100%. The formation of both single and numerous apoptotic bodies inside the cell was observed in tumor cells. Under the same conditions, a smaller number of apoptotic cells (14–16%) were found in spleen cells (splenocytes) of healthy mice, probably due to the action on only cells ready for apoptosis. Previously, we discovered the effect of anphen sodium on antiapoptotic proteins of the Bcl-2 family and hypothesized that this compound leads to apoptosis by the mitochondrial pathway. Since H 2 O 2 at low concentrations can act as a secondary messenger and stimulate the external pathway of apoptosis, it is supposed that the joint action of H 2 O 2 and anphen sodium leads to increased apoptosis by activating the mitochondrial and extrinsic signaling pathways.

The possibilities of using natural resveratrol polyphenol as an adaptogen were investigated by means of atomic force microscopy (AFM) and fluorometry. The study was carried out on mitochondria of pea seedlings (Pisum sativum L., Flora 2 variety). The protective properties of the drug were studied using models of mitochondrial “aging” and water deficiency. Incubation of isolated mitochondria in a hypotonic medium (the aging process) activated lipid peroxidation (POL): the fluorescence intensity of POL products increased compared to the control and led to an increase in the size of AFM images of mitochondria, which indicated swelling of the organelles. Resveratrol at a concentration of 10−6 M reduced the fluorescence intensity of lipid peroxidation products almost to control values, as well as preventing mitochondrial swelling. In vivo experiments were carried out in a water deficiency model. Pea seeds previously soaked in water or treated with resveratrol were subjected to water deficiency. Water-deficiency conditions caused an increase in lipid peroxidation of the mitochondrial membranes of pea seedlings, led to swelling of mitochondria and inhibition of the biogenesis of these organelles as compared to the control. Resveratrol treatment of pea seeds (3 × 10−4 M) reduced the number of swollen mitochondria in relation to the control, activated the biogenesis of these organelles, and normalized POL. It was hypothesized that the protective effect of the studied drug is due its antioxidant properties and AFM and fluorometry methods can be used to screen the protective properties of biologically active substances.

The antineoplastic properties of an antioxidant, sodium 2-carboxy-2-(N-acetylamino)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, are studied on an ascites tumor, Sarcoma 37, and on a solid tumor, Lewis carcinoma. A full suppression of Sarcoma 37 and an intensification of apoptosis (a decrease in the level of Bcl-2 antiapoptotic protein) in the cells of Lewis carcinoma are observed for intraperitoneal injection of the preparation. The structure of the complexes of sodium 2-carboxy-2-(N-acetylamino)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate with peptides is calculated using quantum chemistry. Quantitative data are obtained which characterize the intra- and intermolecular interactions of the above components in nanoassociates.