The laws of delayed photohaemolysis sensitized by chlorin e6.

The relationships between the rate of post-irradiated photohaemolysis sensitized by chlorin e6 and parameters such as the light fluence (time of irradiation) and sensitizer concentration were studied. On the basis of the single-parametric approach proposed by Valenzeno and Pooler, it was found that the haemolytic rate varies with the square of both the light fluence and the sensitizer concentration. Thus it can be concluded that, in a single erythrocyte lesion, two chlorin e6 molecules participate, each absorbing one photon. The possibility of suppression of post-irradiation haemolysis was also studied using the lipophilic antioxidant, butylated hydroxytoluene (BHT), and scavengers of 1O2, O2.- and HO. radicals. It was found that BHT inhibits, to a considerable extent, the post-irradiation lysis of cells, by about a factor of 2.5 at a BHT concentration of 9 microM. The addition to the medium of NaN3 (a scavenger of 1O2), superoxide dismutase (a scavenger of O2.- radicals), ethanol and D-mannitol (scavengers of HO. radicals), when irradiation was interrupted, did not produce a marked influence on the kinetics of subsequent haemolysis. On the basis of the results obtained, the nature of erythrocyte targets, which are crucial for the photodynamic effect of chlorin e6, is discussed.

Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo.

Several parameters of chlorin e6 and its derivative chlorin e6 ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin e6 ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin e6 ethylenediamide is a much better tumour localizer than chlorin e6. The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.

[The porphyrin content of the erythrocytes in cancer patients]. / Soderzhanie porfirinov v éritrotsitakh u onkologicheskikh bol'nykh.

Levels of copro- and protoporphyrin in red blood cells obtained from patients with tumors of the stomach, large bowel and thyroid are discussed. The levels of both porphyrins proved markedly increased in patients with gastric and intestinal cancer but were nearly normal in cases of benign tumor of various sites and thyroid cancer.

[The use of porphyrins for assessing the effect of low doses of ionizing radiation on the human body]. / Ispol'zovanie porfirinov dlia otsenki vliianiia malykh doz ioniziruiushchego izlucheniia na organizm cheloveka.

Data on porphyrin metabolism disturbances under the action of ionizing radiation on the human body are presented. The studies are based on estimating the quantitative content of coproporphyrin in the diurnal urine diuresis of persons irradiated as a result of the Chernobyl accident. The results of examining the population of a number of the zones of close control in Mogilev and Gomel Provinces are considered. Unlike the presumably healthy subjects, many people of this group show a significant decrease of porphyrin excretion with urine. The discovered sensitivity to the action of radiation is individual. A preliminary estimation of the porphyrin test depending on the degree of morphological changes in the gastric mucosa was made during an all-round clinical examination of the irradiated subjects.

Chlorin e6-liposome interaction. Investigation by the methods of fluorescence spectroscopy and inductive resonance energy transfer.

On the basis of spectral fluorescence and polarization measurements and results obtained on the luminescence quenching of the membrane fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) by incorporated chlorin e6 (chl e6) molecules, it is shown that the interaction of the water-soluble pigment with smaller unilamellar lipid vesicles occurs by a mechanism of partition between the aqueous and lipid phases (partition coefficient Kp = 6.7 x 10(3) and provides rigid fixing of chl e6 monomers at the boundary between the polar and non-polar parts of the lipid membrane. In terms of inductive resonance electronic excitation energy transfer between DPH and chl e6 (R0 = 36.2 A), we have analysed data on DPH fluorescence quenching under different conditions of chl e6 localization in the lipid bilayer and have concluded that the incorporation of the pigment molecules into the vesicles from the aqueous phase occurs mainly into the external monolayer.

[The distribution of chlorine e6 and its derivatives in HeLa cells studied using luminescence microscopy]. / Issledovanie raspredelenie khlorina e6 i ego proizvodnykh v kletkakh HeLa metodom liuminestsentnoi mikroskopii.

The influence of the chemical structure of porphyrin pigments on their accumulation and localization in HeLa cells has been examined by the scanning fluorescence microphotometry. It has been found that the replacement of carboxyl groups of chlorine e6 for methyl and amino groups has no influence on the pigment distributions in cells. All the pigments are bound by cell membrane structures. The chemical modification of chlorine e6 structure is essential for the ability of pigment to be accumulated by cells that can be used to increase the efficiency of cancer phototherapy. The charge and hydrophobic properties of pigment molecules are of great importance for accumulating porphyrin sensitizers by cells.

[Singlet oxygen and photodestruction of erythrocyte membranes, sensitized by chlorin e6]. / Singletnyi kislorod i fotodestruktsiia éritrotsitarnykh membran, sensibilizirovannaia khlorinom e6.

By using scavengers of active oxygen forms (AOF) the role of 1O2, O2- and HO in photodestruction of protein and lipid components of erythrocyte membranes and also in photohemolysis of erythrocytes sensitized by chlorine e 6 were studied. It was found that 1O2 plays the dominant role in these processes, but contribution of other AOF, O2- in particular, can not be fully excluded.

[Characteristics of complex-formation of chlorine e6 with human and bovine serum albumins]. / Zakonomernosti kompleksoobrazovaniia khlorina e6 s syvorotochnymi al'buminami cheloveka i byka.

Pigment-protein complexes of chlorin e6 (Chl e6) with human (HSA) and bovine serum albumines (BSA) have been investigated by spectral-luminescent methods. Fluorescence quenching of tryptophan residues caused by the inductive-resonance energy transfer to pigment molecules and the rise of the polarization degree of Chl e6 emission were observed upon incorporation of Chl e6 in the protein globula. The obtained data on spectral-energetic parameters of protein tryptophanyls and Chl e6 permitted us to calculate the energy transfer critical distances R0 in complexes of Chl e6 with HSA (R0 = 32 A) and BSA (R0 = 35A). The binding constants (K) and the number of binding sites (N) of Chl e6 with HSA and BSA have been obtained from the experiments on tryptophanyl fluorescence quenching of the investigated proteins and polarization measurements of pigment emission (KHSA = 1.2.10(6) mole-1, KBSA = 3.6.10(6) mole-1, NHSA = NBSA = 1). On the basis of the measured values of electronic excitation energy transfer efficiency (phi greater than or equal to 99%) the average distances between the protein chromophores and the incorporated Chl e6 molecules have been calculated (RHSA = 15-17 A, RBSA = 16.5-18 A). The questions connected with pigment localization sites in the protein globula and specific features of pigment-protein interaction are discussed.

[Photodynamic effect of chlorine e6 on erythrocyte membranes]. / Fotodinamicheskoe deistvie khlorina e6 na membrany éritrotsitov.

Photodestruction of erythrocyte membranes sensitized by water soluble chlorophyll derivative chlorine e6 (Chl e6) was studied. It has been determined that light irradiation of erythrocyte ghosts with wave length lambda-660 nm in the presence of Chl e6 caused in protein and lipid components of the membrane deep destructive changes which were expressed in cross-linking of the membrane polypeptides and accumulating in the membrane products of peroxidation of unsaturated fatty acids residues, in phospholipids mainly. It has been shown that these processes were realized only in the presence of molecular oxygen and consequently possess pure photodynamic character.

[Photosensitization by chlorine and porphyrin causing structural damage of erythrocytes]. / Fotosensibilizirovannye khlorinami i porfirinami structurnye povrezhdeniia éritrotsitov.

Kinetics of hemolysis of human erythrocytes photosensitized by porphyrin and chlorin derivatives was investigated by small angle light scattering method. The compounds used were arranged in the following order of photosensitizing activity decrease: ethylendiamide of chlorine e6, chlorine e6 dimethylester chlorine e6 haematoporphyrin diacetate tetracarboxyphenylporphyn. The substances having a greater number of binding sites with liposomes (chlorine e6 dimethylester) or with albumin (ethylendiamide of chlorine e6) showed the greatest photohemolytic activity. The photohemolytic activity of porphyrins was suggested to depend on the number of the pigment molecules bound by erythrocytes membranes.

[Erythrocyte photohemolysis sensitized by chlorin e6]. / Fotogemoliz éritrotsitov, sensibilizirovannyi khlorinom e6.

Hemolysis of human erythrocytes photosensitized by chlorophyll derivative--chlorin e6 has been studied. This process was found to be oxygen-dependent. High photodynamic activity of chlorin e6 is explained both by its specific binding with cells and by its photophysics parameters.

[Mixed aggregation of pigments with participation of bacteriochlorophyll]. / Smeshannaia agregatsiia pigmentov s uchastiem bakteriokhlorofilla.

In binar mixture of solvents (dioxane--water, 1 : 4) the spectral-luminescent investigation of the regularities of mixed association and the energetic interaction between molecules of bacteriochlorophyll (BChl) and its analogs (chlorophyll a--Chl, protochlorophyll--PChl, 4-vinyl-protochlorophyll--4VPChl) under variation of donor-acceptor ratio (0,001 less than or equal to CD/CA less than or equal to 1) in the polymer complex was conducted. The essential role of the elementary cell structure of the mixed associate was discovered: the associate of individual BChl did not possess measurable fluorescence whereas the introduction of PChl, 4VPChl and Chl admixture (approximately greater than 1%) into BChl aggregate resulted in the appearance of a BChl fluorescent ability. It was suggested that admixture and BChl molecules formed dimer cells in which the excitation nonradiative deactivation probability was strongly decreased in comparison with individual BChl aggregate. These cells are the centres of mixed associates emission. It was shown that under conditions of effective energy migration in the system of nearly arranged centres with a high degree of orientation the excitation trapping probability strongly depended on the resonance conditions for transition density--transition density interaction between partners of aggregation.

[Spectral-luminescent properties of separate aggregated forms of pigments in solutions]. / Spektral'no-liuminestsentnye svoistva otdel'nykh agregirovannykh form pigmentov v rastvorakh.

The aggregated forms of protochlorophyll a (PChl), 4-vinyl-protochlorophyll a (4VPChl) and chlorophyll a (Chl) in binar mixtures of dioxane-water was investigated. The aggregates are oligomer particles of pigment molecules having considerable degrees of fluorescence polarisation which points to their ordered structure. Two types of fluorescent associates I and II have been discovered. By its electronic spectrum I is similar to the molecular pigment but has smaller quantum yield B congruent to 10(-2) and the lifetime of fluorescence tau congruent to 3--4.10(-9) sec. Associates of type I spontaneously transform into associates of type II which have narrower bands of electronic spectrum than the monomer, very low B congruent to 10(-3)--10(-4) and tau congruent to 2--4.10(-10) sec. In contrast with the decreasing of B and the band intensities in CD spectrum conditioned by rotational strength increase by factor of 100 under transformation from I to II. It follows from these data that the antibatic connection possibly exists between excitation deactivation in associates and their optical activity. From data on splitting the associate CD spectra of type I and II the excitation interaction energy V12 congruent to 75 cm-1 was estimated. The reasons of increasing of rotational strength under transformation from I to II and the overturn of the sign dependence of bands in CD spectrum in some cases are discussed.

[Mechanism of photoregeneration of reduced porphyrin in the presence of oxygen]. / Mekhanizm fotoreganeratsii vosstanovlennogo porfirina v prisutstvii kisloroda.

Photooxidation of reduced forms of porphyrines (IIH2) was studied. It has been shown that photooxidation of IIH2 is sensitized by initial porphyrine formed in the course of the reaction. Singlet oxygen serves as the basic oxidizer. It is also informed that the search of metastable states of photoreduced anion forms of the pigments by means of flashphotolysis turned to be unsuccessful.

[Intermediate reduction products of porphyrins and their structure]. / Promezhutochnye produkty reaktsii vosstanovleniia porfirinov i ikh stroenie.

It was shown on the basis of NMR-H spectra that the addition of proton to dianions of Zn-octaethylchlorine, Zn-octaethylporphin, and Zn-monoasaethioporphyrin results in the formation of products with the structure of alpha-dihydroflorine, alpha-florine, and gamma-florine respectively. This indicates that electron density greatly increases when the anions are formed in the centres which had a decreased density in the initial molecule.

[Relationship between chlorophyll concentration and the energy reaction between protochlorophyll and chlorophyll in mixed associations]. / Izuchenie énergeticheskogo vzaimodeistviia mezhdu protokhlorofillom i khlorofillom v smeshannykh assotsiatakh v zavisimosti ot kontsentratsii khlorofilla.

In the paper results are presented of investigation of protochlorophyll (PChl) and chlorophyll (Chl) mixed associations and of interaction between them within the polymer molecular complex, which forms in mixture of water-dioxane (1 : 4). The initial PChl concentration in all solutions was constant (CPChl = 1 . 10(-5) m/l), and Chl concentration varied from 1.10(-8) m/l up to 2.10(-5) m/l. It is shown that with the rise of Chl proportion in the mixed aggregate the rearrangement of both donor (PChl) and acceptor (Chl) components of complex takes place. The luminescence quenching of PChl and the sensitization of Chl emission in mixed pigment associates were investigated of different Chl levels and the evaluation of efficiency of intracomplex electronic excitation energy transfer, determined from quenching and sensibilization, was performed. Similar dependence of energy transfer effectiveness on Chl concentration, determined by the two above-mentioned methods shows that the excitation migration in an associate takes place without losses. An analysis of results permits to conclude that a small trapping efficiency of PChl excitation by the acceptor part of the complex may be connected with the existence of the prerelaxation reverse energy transfer from Chl to PChl in mixed pigment associates. On the basis of the obtained data a mechanism of energy transfer from protochlorophyllide to chlorophyllide in etiolated leaves and homogenates is discussed.