M. G. Strakhovskaya

THE ROLE OF MEMBRANE-BOUND PORPHYRIN-TYPE COMPOUND AS ENDOGENOUS SENSITIZER IN PHOTODYNAMIC DAMAGE TO YEAST PLASMA MEMBRANES

The effect of visible light (400-600 nm) on Candida guilliermondii and Saccharomyces cerevisiae was studied, and irradiation fluences killing the yeast cells without exogenous sensitizers were determined. The lethal effects are strongly oxygen-dependent, suggesting the involvement of photodynamic reactions mediated by endogenous sensitizer(s). Repair-deficient strains of S. cerevisiae show the same photosensitivity as the wild-type strain indicating that visible light does not photosensitize repairable DNA lesions. As was demonstrated using the microfluorometric method with the fluorochrome primulin, photodestruction of plasma membrane permeability barriers is important for yeast cell lethality. Visible light at cell-killing fluences induces lipid peroxidation in plasma membrane ghosts isolated from C. guilliermondii. Data obtained suggest the important role of singlet oxygen photogenerated by endogenous sensitizer(s) in initiating oxidative reactions. A spectrofluorometric analysis of the plasma membrane ghosts revealed one compound fluorescent in the visible spectral region at 683 nm. Its fluorescence excitation and absorption spectra have structures typical for porphyrins. The plasma membrane-bound porphyrin-like compound is different in some fluorescence properties from mitochondrial porphyrins. Estimation of its amount gives a value of 0.1 nmol porphyrin per milligram of protein of the plasma membrane ghosts. This porphyrin-type compound is considered to be the most probable candidate for the role of the sensitizer in photodynamic damage to yeast plasma membrane and cell inactivation by visible light.

Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: Its role in photodynamic inactivation

The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca2+ and Mg2+ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.

Protection of Saccharomyces cerevisiae against Oxidative and Radiation-Caused Damage by Alkylhydroxybenzenes

The effects of C7-alkylhydroxybenzene (С7-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under γ irradiation of cell suspensions using doses of 10–150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensitized with chlorin e6 (10 μg/l). C7-AHB was found to exert a protective effect. The addition of 0.05–0.16 vol % of C7-AHB to cell suspensions 30 min before irradiation protected yeast cells from γ radiation (50 krad). The protective effect of C7-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C7-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C7-AHB demonstrated that the protective mechanism of С7-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial С7-AHB are not related to its chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.

New heterogeneous photosensitizers with phthalocyanine molecules covalently linked to aminopropyl silica gel

New heterogeneous photosensitizers were synthesized, in which phthalocyanines of zinc and aluminum, tetrasubstituted at non-peripheral positions with modified thiophenyl groups, were grafted to aminopropyl silica gel. The absorption and fluorescence spectra, and the quantum yields of fluorescence and photosensitized singlet oxygen generation were estimated for aqueous suspensions of these sensitizers. It is shown that upon photoexcitation, silica gel-bound phthalocyanines produce singlet oxygen and display photobactericidal activity against bacteria E. coli.

NEAR‐UV ACTIVATION OF ENZYMATIC CONVERSION OF 5‐HYDROXYTRYPTOPHAN TO SEROTONIN

Abstract— Near‐UV (337 nm) photoactivation of the 5‐hydroxytryptophan decarboxylation reaction producing serotonin has been observed. The photoactivation effect was investigated as a function of fluence rate and fluence, and pH. Photoactivation of decarboxylase activity was found to occur at nearly neutral pH values (low activity of the enzyme in the dark). The findings indicate that the effect of light is similar to a pH shift toward the acid region, which causes the enzyme conversion from the inactive to active form. Pyridoxal phosphate, the decarboxylase cofactor, in the form of an adduct absorbing at 330–340 nm, is suggested as a candidate for the role of the photoactive chromophore of decarboxylase.

Effects of the degree of substitution on the physicochemical properties and photodynamic activity of zinc and aluminum phthalocyanine polycations

A series of zinc and aluminum phthalocyanines containing 3–8 pyridiniomethyl or cholinyl substituents on average were synthesized. As the number of cation substituents increased, in aqueous solutions, the aggregation ability of phthalocyanines decreased, while the quantum yields of fluorescence and singlet oxygen generation increased. The photodynamic inactivation of coliform bacteria sensitized by zinc and aluminum phthalocyanine polycations with an increase in the substitution degree became more effective.

The Photodynamic Inactivation of the Yeast Candida guilliermondii in the Presence of Photodithazine

Photodithazine, a glucosamine salt of chlorin e6, is highly effective in sensitization of Candida guilliermondii cells to visible light. The sensitizing effect of photodithazine was found to be related to free or cell surface–bound molecules of this dye. Sodium azide (a singlet oxygen quencher) and propyl gallate (an inhibitor of lipid peroxidation) protected yeast cells from the photodithazine-enhanced photoinactivation.

Fungicidal activity of khlorin photosensitizers.

In recent years, there has been increasing interest of researchers in new methods of control of pathogenic microorganisms. This was due to a significant increase in the number of strains resistant to conventional antimicrobial preparations [1, 2]. Photodynamic therapy (PDT) is one of these new methodological approaches. PDT includes a combination of three essential components: a light-absorbing substance (photosensitizer), light, and oxygen [3]. The principle of the PDT method is that the photoexcited state of the photosensitizer molecule mediates the generation of active oxygen species. Active oxygen species themselves induce the processes of oxidative destruction, thereby causing structural disintegration and inhibition of the functional activity of subcellular structures. As a result, inactivation of cells is observed [3, 4]. Until recent time, PDT has been used for treating some forms of skin cancer [5]. On the other hand, in the last decade, there has been significant progress in development of methods of antimicrobial PDT [6, 7].

Biological photoreceptors of light-dependent regulatory processes.

Progress in understanding primary mechanisms of light reception in photoregulatory processes is achieved through discovering new biological photoreceptors, chiefly the regulatory sensors of blue/UV-A light. Among them are LOV domain-containing proteins and DNA photolyase-like cryptochromes, which constitute two widespread groups of photoreceptors that use flavin cofactors (FMN or FAD) as the photoactive chromophores. Bacterial LOV domain modules are connected in photoreceptor proteins with regulatory domains such as diguanylate cyclases/phosphodiesterases, histidine kinases, and DNA-binding domains that are activated by photoconversions of flavin. Identification of red/far-red light sensors in chemotrophic bacteria (bacteriophytochromes) and crystal structures of their photosensor module with bilin chromophore are significant for decoding the mechanisms of phytochrome receptor photoconversion and early step mechanisms of phytochrome-mediated signaling. The only UV-B regulatory photon sensor, UVR8, recently identified in plants, unlike other photoreceptors functions without a prosthetic chromophore: tryptophans of the unique UVR8 protein structure provide a “UV-B antenna”. Our analysis of new data on photosensory properties of the identified photoreceptors in conjunction with their structure opens insight on the influence of the molecular microenvironment on light-induced chromophore reactions, the mechanisms by which the photoactivated chromophores trigger conformational changes in the surrounding protein structure, and structural bases of propagation of these changes to the interacting effector domains/proteins.

Photoquenching of the Bioluminescence of the Genetically Engineered Escherichia coli TG1 (pXen7) Strain in the Presence of Photodithazine

The photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain was studied in the presence of the photosensitizer photodithazine, a glucosamine salt of chlorin e6. The photosensitized quenching of the bioluminescence was found to correlate with the colony-forming ability of the strain. The data obtained are discussed from the standpoint of using biosensor luminescent bacterial systems for the assessment of the efficiency of photosensitizers in antimicrobial photochemotherapy.