UROCANIC ACID PHOTOBIOLOGY. PHOTOCYCLOADDITION OF N,N-DIMETHYLTHYMINE TO UROCANIC ACID*

Abstract— Photolysis of N,N-dimethylthymine (DMT) and urocanic acid (UA) gives rise to two isolable cyclobutane adducts. N,N-dimethylthymine also photosensitizes UA E / Z isomerization. Neither phenomenon is affected by a triplet quencher, and ¹DMT* is proposed as the likely reactive species. If so, prior complexation of DMT and UA would be necessary in order for their interaction to be competitive with DMT radiationless decay. These observations are potentially related to the effect of UA on viral DNA described in the following paper.     

Z/E PHOTOISOMERIZATION OF UROCANIC ACID*

The E ? Z photoisomerization of the title compound (UA) (a naturally occurring sunscreen) has been studied in aqueous solution. At a UA concentration of 6mM and using 313nm excitation, φ_E→z= 0.52, φ_Z→E= 0.47 and the photostationary state is 34% E. Under these conditions, loss of UA is minimal. Low energy triplet quenchers fail to impede the isomerization, but the reaction can be induced by several triplet sensitizers. The E_T for UA is estimated to be approximately 55 kcal/mol.     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

PHOTOCHEMICAL DECARBOXYLATION OF AMINO ACIDS IN THE PRESENCE OF METAL IONS

Abstract— An electron-spin-resonance study of the radicals produced by ultraviolet irradiation of amino acids in the presence of transition metal ions at low temperature has been carried out. For the monocarboxylic acids, the photosensitized irradiation resulted in loss of carbon dioxide, which led to the formation of free radicals of the type H₃ŃR₁R₂. When the amino acid contained two carboxylic groups, e.g. aspartic acid, decarboxylation took place at the position remote from the amino group. When the odd electron is localized at the carbon atom β to the amino group, e.g. β-alanine and aspartic acid, it was stable enough to be detected at room temperature.     

STRUCTURE OF UROCANIC ACID PHOTODIMERS

Abstract— U.V. irradiation of trans -urocanic acid frozen in an ice matrix results in trans, trans, trans -2,4-di-imidazolyl-1,3-cyclobutanedicarboxylic acid from pH 8.0 solutions; or cis, trans, cis -3, 4-di-imidazolyl-1, 2-cyclobutanedicarboxylic acid from pH 5.9 solutions.     

PROTEIN ENHANCED INACTIVATION OF VIRAL DNA BY UV RADIATION

Abstract Viral DNA was irradiated in the presence of proteins and assayed for loss of biological activity. Several proteins were found to enhance the inactivation of transfecting ability by UV radiation. The sensitization caused by a particular protein depends upon its amino acid composition. Experiments done at low molar ratios of protein to DNA indicate that a protein-induced lesion could be a highly efficient means of inactivation.     

KINETIC FORMALDEHYDE ANALYSIS OF DNA ULTRAVIOLET IRRADIATED IN THE PRESENCE OF SILVER IONS*

Abstract— DNA from Escherichia coli was irradiated at 254 nm in the presence of silver in order to preferentially enhance the rate of formation of pyrimidine-dimer damage over nondimer damage. The irradiated DNA was treated with formaldehyde in order to measure the unwinding velocity of the defects associated with the pyrimidine dimers. This velocity was found to be 0.18 base pairs/min per pyrimidine dimer, which is nearly 8 times less than that found for a double-strand break (1.37 base pairs/min) obtained by use of sheared DNA whose size was determined by electron microscopy. The rate of reaction of the DNA with formaldehyde varied linearly with the pyrimidine dimer concentration and showed no inflection due to clustering. Treatment of irradiated DNA with UV endonuclease enhanced the formaldehyde reaction by ? 7-fold, consistent with the conversion of a dimer into the faster-reacting defect associated with a single-strand break. These results indicate that the distribution of dimers in DNA is random and not clustered, and that previous interpretations of clustering were based on the false assumption that dimer and chain break defects unwind with similar velocities when treated with formaldehyde.     

PHOTOSENSITIZED INACTTVATION OF INFECTIOUS DNA BY UROCANIC ACID, INDOLEACRYLIC ACID AND RHODIUM COMPLEXES

Naked, infectious single-stranded (ss) and double-stranded (ds) DNA from phages SI3 and G4 were irradiated with 308 nm UV radiation in the absence and presence of several photobiologically active compounds: E - and Z -urocanic acid ( E - and Z -UA), their methyl esters ( E - and Z -MU), E - and Z-indoleacrylic acid ( E - and Z -IA), cis -dichlorobis(1,10-phenanthroline)rhodium(III) chloride (cDCBPR) and tris(1,10-phenanthroline)rhodium (III) perchlorate (TPR). E -urocanic acid protects against cyclobutane pyrimidine dimer formation in ssDNA but concomitantly photosensitizes the formation of other lesions that inactivate ssDNA. Z -urocanic acid also protects ssDNA against such dimerization but without the associated sensitized damage. The methyl ester isomers behave similarly. There is no such differential activity observed for the IA isomers, both of which sensitize the inactivation of ssDNA. Photostationary state mixtures of both UA and IA efficiently sensitize the inactivation of dsDNA, and cDCBPR strongly protects ssDNA from UV damage, while TPR is a significant sensitizer. Both of these metal complexes sensitize the inactivation of dsDNA slightly. For all compounds, cyclobutane pyrimidine dimers were the predominant lethal lesions produced by sensitization of the dsDNA, but they were not the major lethal lesions created by sensitization of the ssDNA. In the case of dsDNA, both UA and IA created pyrimidine dimers with a high degree of potential for mutagenesis, as determined by an assay that monitors the frequency of mutations following the spontaneous deamination of cytosine in photodimers.     

UV INACTIVATION OF ENZYMES IN SUPRAMOLECULAR COMPLEXES OF BIOLOGICAL MEMBRANES. THE PHENOMENON OF PHOTOCHEMICAL ALLOTOPY

Abstract. The photosensitivity of erythrocyte acetylcholinesterase (AChE) is different in its free and membrane-bound states. The modification of the structure of membraneous lipids by phospholipases A₂, C and D or by cholesterol depletion is accompanied by a change in AChE photosensitivity. UV light was demonstrated to induce cooperative structural transitions in the erythrocyte membrane. This follows from the data obtained by circular dichroism and solubilization in detergents. In contrast to free AChE, UV light acts on the membraneous enzyme as a mixed inhibitor (simultaneous change in V _max and K _m). The anomalous behaviour of membrane-bound enzyme, termed the phenomenon of photochemical allotopy, is associated with a modification of the structure within the microenviron-ment of the residual AChE. The phenomenon depends on membrane integrity, and disappears after treatment of erythrocyte ghosts with ultrasound, trypsin, phospholipases and neuraminidase and remains unchanged in cholesterol-depleted membranes. The nature and localization of events responsible for this phenomenon are discussed.     

HETEROGENEITY OF THE PHOTOCHEMICAL CENTERS IN SYSTEM II OF CHLOROPLASTS*

Abstract— In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts of algae and‘ higher plants the area over the fluorescence induction curve increases with biphasic first order kinetics (Melis and Homann, 1975). Two possibilities are considered to explain the biphasic nature of the area growth. The first is a sequential double reduction of the primary electron acceptor in system II while the second envisages a heterogeneity of its photochemical centers. The kinetic properties of the area growth after firing a single saturating flash proved to be incompatible with the predictions of the “sequential double reduction” model. This conclusion was corroborated by results obtained from a kinetic analysis of the area restoration process in the dark, and an analysis of the partially restored areas. Assuming an existence of a heterogeneous pool of photochemical centers, the growth of the area over the fluorescence curve could be further analyzed to yield two components, a fast a-component, and a relatively slow β-component. The kinetic characteristics of these components, and the effect of a short saturating flash on their respective size, led to the conclusion that one type of photochemical center had a faster recombination rate of the photochemically separated charges and was less efficient in trapping excitation energy.     

PAIRWISE RECOMBINATION IN THE DECAY OF PHOTOCHEMICAL HYDRATED ELECTRONS*

Abstract— The decays of hydrated electrons generated by 265 nm laser flash photolysis of aqueous I^-, tyrosine, tryptophan, lysozyrne. and ribonuclease have been analyzed by numerical computing. Homogeneous reactions do not explain the data without e^- _aq—radical back reactions significantly faster than diffusion limited. The results are explained by postulating pairwise recombination persists at times where conventional diffusion theory predicts randomization, particularly at low intensities and in the absence of effective e^- _aq scavengers. The physical mechanisms responsible for these effects are considered.     

LANGMUIR MONOLAYERS OF TWO-HEADED SURFACTANTS CONTAINING THE UROCANIC ACID MOIETY

The properties of Langmuir layers made up of three bola-ampbiphiles having the general structure UA-C_n-UA, where UA denotes urocanic acid and C_n is an alkyl chain of varying length (n = 8, 12 and 16) and of a monopolar amphiphile (UAC₁₂) were investigated. The area per molecule at the air-water interface was measured. Moreover, a modeling study was performed to visualize the conformation of these compounds at the air / water interface. The pressure-area isotherm and the molecular modeling study indicate that a folded conformation is possible only for the UAC₁₆UA bola-amphiphile. This result could be connected to the ability of UAC₁₆UA to form micelles.     

PHOTOSENSITIZED INACTIVATION OF DNA BY MONOCHROMATIC 334-nm RADIATION IN THE PRESENCE OF 2-THIOURACIL: GENETIC ACTIVITY AND BACKBONE BREAKS

Abstract Monochromatic 334-nm radiation delivered under aerobic conditions inactivates the genetic activity (ability to transform auxotrophic recipient cells to nutritional prototrophy) of isolated transforming Bacillus subtilis DNA. The presence of superoxide dismutase (SOD), catalase, and mannitol reduces the 334-nm inactivation. The rate of inactivation of the genetic activity by 334-nm radiation is enhanced fivefold by the sensitizer 2-thiouracil (s²Ura). This enhancement is substantially reversed when the irradiations are performed in the presence of mannitol, and, to a lesser extent, SOD. Catalase slightly reduces the s²Ura enhancement of 334-nm inactivation of transforming activity. Backbone breaks induced in the same DNA by aerobic 334-nm radiation were also enhanced markedly by the presence of s²Ura; this enhancement was reversed by the presence of mannitol and, to a lesser extent, SOD during irradiation. Catalase had no effect upon s²Ura-enhanced, 334-nm-induced SSBs. Whereas DNA breakage may be responsible for a portion of the inactivation of the DNA by the photosensitized reaction between s²-Ura and 334-nm radiation, it is not the only inactivating lesion, because the yield of SSBs per lethal hit per unit length of DNA is not constant for all the irradiation conditions studied. The results support a complex role for active oxygen species in inactivation of transforming activity and DNA breakage by s²Ura-enhanced 334-nm radiation. They are also consistent with the formation of superoxide anion, hydroxyl radical, and possibly also singlet molecular oxygen, generated from ground-state molecular oxygen by reactive s²Ura in both Type I and II reactions.     

反相高效液相色谱测定在乙醇酸和草酸存在下的乙醛酸含量

采用反相高效液相色谱分离测定了乙醇酸和草酸共存下乙醛酸的含量。色谱柱为Hy-persil ODS-C18柱(4.6 mm×100 mm,5μm),流动相为乙腈-水,检测波长为210 nm。探讨了流动相的pH及其配比对分离度和灵敏度的影响。以峰面积对乙醛酸浓度(g.L-1)作图所得校正曲线的线性回归方程为Y=1.934×103X+0.067×103(r=0.999 9),方法的检出限(2S/N)为5×10-7g.L-1。方法的测定回收率为98.1%~100.4%,相对标准偏差为0.4%。     

SEDIMENTATION ANALYSIS OF DNA PHOTOOXIDIZED IN THE PRESENCE OF THIOPYRONINE

Abstract. Illumination of single-stranded φ×174 phage DNA with visible lightλ > 500 nm) in the presence of the sensitizer thiopyronine results in both chain scissions detectable by velocity sedimentation in neutral medium and in alkali-labile bonds which yield secondary strand breaks after alkaline treatment. Compared with the generation of primary strand breaks, the formation of alkali-labile sites seems to be the predominant reaction.
Photodynamic treatment of homogeneous double-stranded Ta, phage DNA leads to changes in the overall conformation of DNA as revealed by an initial increase of the sedimentation coefficient. The simultaneous occurrence of different effects (decrease of molecular weight, increase of effective DNA flexibility) is particularly evident from changes in the sedimentation coefficient distribution. The fact that both processes influence the sedimentation behaviour questions the common procedure of calculating double-strand break numbers from sedimentation coefficient distributions on the basis of s#-M relations which are valid for intact DNA only.
Photooxidized double-stranded DNA exhibits an increased sensitivity against shear forces.     

PHOTOCLEAVAGE OF DNA IN THE PRESENCE OF SYNTHETIC WATER-SOLUBLE PORPHYRINS

In the presence of oxygen and visible light, various synthetic water-soluble porphyrins cleave pBR 322 plasmid supercoiled DNA (form I) producing relaxed (form II) and linear (form III) DNA corresponding to single-strand and double-strand breaks respectively. Large variations are observed in the efficiency of the porphyrins containing a diamagnetic metal or no metal at all. Singlet oxygen (¹O₂) seems to be involved in the mechanism of cleavage consistent with the inhibitory effect of the azide anion, N^–₃. The higher efficiency of cationic porphyrins (as compared to anionic ones) is due to their greater affinity for DNA as shown by experiments carried out at either high ionic strength or in the presence of the surfactant, sodium dodecyl sulfate.     

ROLE OF DYE MOLECULES REMAINING OUTSIDE THE CELL DURING PHOTODYNAMIC INACTIVATION OF ESCHERICHIA COLI IN THE PRESENCE OF ACRIFLAVINE

Abstract— Photodynamic inactivation of cells is caused by damage to the regions proximal to the cell envelope or to the DNA via a singlet oxygen mechanism. For penetrating dyes the possibility of either type of damage remains. The contribution of a penetrating dye. acriflavine. remaining outside E. coli B/r cells during irradiation. towards photodynamic inactivation was investigated. It was found that this contribution was either nil or negligible.