INVESTIGATION OF DAMAGE TO FOREST BY EPR SPECTROSCOPY in vivo

Spruce needles collected from several trees of the Black Forest were investigaled by EPR spectroscopy. These needles show in the g = 2.00 region a signal II_S(Tyr D ₊) and a light-induced signal I(P700₊) and a Mn₂₊ hyperfine structure which superimposes the other absorptions. Difference spectra, light minus dark, partly eliminate the manganese hyperfine structure, and P700₊ can be observed. By comparison of these EPR signals with those of spinach chloroplast or thylakoid membranes described in the literature, significant deviations were observed, whereas several trees grown in the vicinity of Tubingen exhibit the well known D₊ and P700₊ EPR spectra. After treatment of branches of these 'normal' trees with herbicides like Amitrol and Roundup or chemicals like toluene or trichlormethane the EPR signals obtained are comparable with those observed with needles of the Black Forest.     

PROFLAVINE PHOTOSENSITIZATION OF DOUBLE AND SINGLE STRANDED DNA: EPR STUDY OF THE EFFECT OF VARIOUS SULPHUR-CONTAINING SUBSTANCES

Abstract—The proflavine-mediated photosensitization of both double-stranded calf thymus and single-stranded DNA from bacteriophage φ× 174 was followed in terms of the induction of free radicals in frozen solutions by EPR measurements in the presence of oxygen. The effect of the addition of various sulphur-containing substances to the proflavine DNA mixtures was studied and quantitatively expressed for definite amounts of proflavine bound to both DNAs.
Upon irradiation with visible light, RS radicals were observed in the presence of these sulphur-containing substances. Some of them caused a decrease in the amount of peroxide radicals normally induced in the photosensitized DNA. This decrease appeared to be linked to the property of the compounds to interact with DNA in such a way that they modify the binding of proflavine to DNA.
Cysteine, cysteamine, cystamine and cystine, protect calf thymus DNA against the "proflavine and light" free radical induction by 55, 58, 64 ahd 62% respectively. φ× 174 single-stranded DNA is also protected by the addition of cysteamine and cystamine, although to a lesser extent than calf thymus DNA (35%). These protection factors were measured under conditions where strong binding of the dye to the DNA was predominant.     

INTERACTION BETWEEN PROFLAVINE AND GUANOSINE 5'-PHOSPHATE: IMPORTANCE OF PHOTOEXCITATION

Abstract— The stoichiometry of the complex formed between the mutagenic drug proflavine and the nucleotide guanosine 5'-phosphate has been demonstrated to be 1:1. Its association constant at room temperature is found to increase from 310 M ^-1 when proflavine is in its ground electronic state, to 1550 M ^-1, when proflavine is in its first excited singlet state. Thus, light absorbed by the drug alters its reactivity which, in turn, results in an appreciable increase in its ability to bind to the nucleotide. The enthalpy and the entropy of the ground state proflavine-guanosine 5'-phosphate complex formation are -12.6 kJ/mol (-3 kcal/mol) and -6.7 J/mol/deg (-1.6 cal/mol/deg), respectively. The implications of these findings concerning the proflavine-DNA interaction as well as the possible importance of electronic excitaton in acridine mutagenesis are discussed. An appraisal of the methodology for analyzing fluorescence quenching data is also given.     

EFFICIENCY OF PHOTOAFFINITY LABELING DNA HOMOPOLYMERS AND COPOLYMERS WITH ETHIDIUM MONOAZIDE *

Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently-linked following photoactivation at the same drug/DNA input ratio. There were significant sequence-related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent-adduct formation decreased in the order poly(dG-dC)^. poly(dG-dC)> poly-(dG). poly(dC)poly(dA-dT). poly(dA-dT)poly(dA). poly(dT). Ethidium monoazide was about 2-fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G-C pairs than the A-T base counterparts. In low ionic buffers (0.015 M Na⁺), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40-fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.     

SYNTHESIS AND CHARACTERIZATION OF WELL-DEFINED BLOCK COPOLYMERS CONTAINING PENDANT, SELF-COMPLEMENTARY QUADRUPLE HYDROGEN BONDING SITES

The title block copolymer (defined as PSUEA) containing pendant,self-complementary quadruple hydrogen bonding sites has been prepared successfully by three steps.First,poly(styrene-b-2-hydroxyethyl acrylate) (defined as PSHEA) was prepared by living radical polymerizing 2-hydroxyethyl acrylate (HEA) initiated by polystyrene (PSt) macro- initiator,which was prepared via nitroxide-mediated polymerization (NMP) technique.After treated by excessive 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (IPD...     

INDUCTION OF FREE RADICALS IN DNA BY PROFLAVINE AND VISIBLE LIGHT: INFLUENCE OF OXYGEN AND IONIC STRENGTH

Abstract —The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine-DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA-proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O₂, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O₂ is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred-fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl₂ between μ# 0–01 and μ=0–1.     

SPECTROSCOPY AND PHOTOSENSITIZATION OF SAPPHYRINS IN SOLUTIONS AND BIOLOGICAL MEMBRANES

Abstract A spectroscopic and photophysical study of three new sapphyrin molecules is presented. The sapphyrin backbone that was derivatized to make them water soluble possesses an absorption band around 700 nm, a desired property for biological photosensitization. We studied the absorption and fluorescence spectra, from which evidence for aggregation in solvents of different polarities was obtained. The extent of aggregation is correlated with the nature of the attached moiety. The absolute quantum yields of singlet oxygen production were measured, with 1,3-diphenyl isobenzofuran as a model target, and were 0.13–0.18 in ethanol. The binding constants to liposomes and to cells were determined spectroscopically and were found to correspond to the hydrophobicities of the compounds, with an additional effect, ascribed to the sugar moiety, which was found in the case of one of the sapphyrins. The efficiency of photodamage to Staphylococcus aureus by sapphyrins and hematoporphyrin was equivalent, on the basis of cells killed per microgram of sensitizer in the incubation mixture.     

SPECTROSCOPY OF POLYENES—IV. ABSORPTION AND EMISSION SPECTRAL PROPERTIES OF POLYENE ALCOHOLS RELATED TO RETINOL AS HOMOLOGUES

A number of polyene alcohols related to retinol as homologues have been examined for their spectral and photophysical properties. The absorption spectra of the polyene alcohols with short polyene chainlength show an intense band system with its maximum at 3000–4000 cm^-1 above that of the main band system. The intensity of this higher-energy band system decreases sharply as the polyene chainlength is increased. All the polyene alcohols fluoresce strongly or moderately strongly at 77 K, the intrinsic fluorescence lifetimes being significantly longer than those expected from the oscillator strengths of the main, low-energy absorption band system. Fluorescence (relatively weak) is also observed at room temperature in the cases of polyene alcohols of long chainlength. A discussion is presented regarding the possible assignments of the various observed absorption band systems, state order and nature of the lowest excited singlet state, and aggregate formation (in 3-methylpentane at 77 K) and its effect on spectral and photodynamical behavior.     

SPECTROSCOPY AND PHOTOCHEMISTRY OF 8-SUBSTITUTED 5-DEAZAFLAVINS

Abstract— Absorption, fluorescence and phosphorescence spectra as well as fluorescence and phosphorescence quantum yields of 8-X-5-deazaflavins (X = C1, NO₂, p -NO₂-C₆H₄, N(CH₃)₂, NH₂, p -NH₂-C₆H₄, p -N(CH₃)₂-C₆H₄-N=N) were determined. It was found that all these data are highly influenced by the substituent at position 8 of the 5-deazaisoalloxazine skeleton. Also the photoreduction of 8-X-5-deazaflavins in the presence of electron donors was studied. It was established that the photoreduction leads to the formation of a 5,5'-dimer and/or a 6,7-dihydro compound. Reduction of the C(6)-C(7) bond is promoted by strong electron-donating substituents and bulky electron donors. 5-Deazaftavins with a reducible substituent at position 8 exhibit reduction of the substituent prior to the reduction of the 5-deazaisoalloxazine skeleton.     

THE SPECTROSCOPY AND PHOTOCHEMISTRY OF NATURALLY OCCURRING AND SYNTHETIC CHROMENES

Abstract— –All naturally occurring and synthetic chromenes studied exhibit photochromism at — 196°C and in some cases at room temperature. Chromenes containing a coumarin moiety have the least photochemistry and also exhibit the strongest emissions. Chromenes containing a hydroxyl group ortho to an acetyl group exhibit solely a phosphorescence. Lapachenole (a 7,8-benzochromene) shows the most rapid and highest percentage conversion to the colored form and no emission. Photocolored form of evodione can be produced by a triplet energy transfer from benzophenone.     

高分子钼铁硫原子簇催化剂的合成反应及其结构表征

以乙烯基吡啶的共聚物为高分子载体,用固相合成的方法,通过多步反应合成了一个高分子钼铁硫原子簇催化剂。用紫外可见光谱和红外光谱分析研究了该催化剂的结构,并说明它具有原子簇结构。本催化剂在KBH_4还原剂存在下,对乙炔加氢反应具有催化活性,可以循环使用。     

SPECTROSCOPY OF POLYENES–III. ABSORPTION AND EMISSION SPECTRAL INVESTIGATION OF POLYENE SCHIFF BASES AND PROTONATED SCHIFF BASES RELATED TO VISUAL PIGMENTS

Abstract— A number of n -butylamine Schiff bases of polyenals related to retinals as homologues and analogues, and their protonated forms, have been studied for absorption and emission spectral properties. The polyene Schiff bases exhibit the same general features in their absorption spectra as those of the parallel polyenals except that the ^lB_u←¹A_g and π*← n singlet transitions are at substantially higher energy in the Schiff bases (the shift being larger for the π *← n transition). The Schiff bases with short polyene chainlength ( n = 2, 3 where n is the number of double bonds including C=N) do not fluoresce or phosphoresce in 3-methylpentane in the temperature range 298–77 K. The Schiff bases with intermediate chainlength ( n = 4, 5) show fluorescence at 77 K with intensity strongly dependent on the nature of solvent. The Schiff bases with relatively long chainlength ( n = 5–7) show strong or moderately strong fluorescence at 77 K and very weak fluorescence at 298 K ( n = 7) with intrinsic radiative lifetimes much longer than those estimated from the oscillator strength of the low-energy, strong absorption band (¹B_u←¹ A_g ). A discussion on the possible state order and nature of the fluorescing state of the various polyene Schiff base systems is presented.     

SYNTHESIS AND CHARACTERIZATION OF NEW OXODIPEROXOVANADATE COMPLEXES CONTAINING COORDINATED AMINOACIDS

Abstract

The study of peroxovanadium compounds has received renewed attention since the discovery of their insulin mimetic properties and of the vanadium bromoperoxidase enzyme which catalyzes the oxidation of halides by hydrogen peroxide. This work presents the synthesis and characterization of three novel oxodiperoxovanadium complexes, K _n [VO(O₂)₂AA] 2H₂O, where AA = L-asparagine(l), L-phenylglycine(2), D, L-homocystine(3). The products were synthesized by the reaction of V₂O₅, with the amino acid and H₂O₂ at room temperature. The compounds obtained are yellow, soluble in water and insoluble in organic solvents. They show remarkable hygroscopic character and are light and temperature sensitive. IR and UV-VIS spectra of the compounds show the typical oxo and diperoxo bands (ν_v = o = 970 cm^?1, ν_vo-o = 870cm^?1, νv-O₂ = 630, 525 cm^?1, ? ?320nm). The ligand bonding properties were determined on the basis of electric conductivity and spectroscopic data (IR, ¹H NMR) as well as elemental analysis.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

A LASER FLASH SPECTROSCOPY STUDY OF THE KINETICS OF ELECTRON TRANSFER FROM SPINACH PHOTOSYSTEM I TO SPINACH AND ALGAL FERREDOXINS

Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from P700 in Photosystem I (PSI)-enriched particles from spinach to the ferredoxins from spinach and the green alga Monoraphidium braunii. Very similar behavior for the interaction of both ferredoxins with the PSI complex was observed, although the algal ferredoxin appears to be slightly more effective as an electron acceptor. For both proteins, a non-linear protein concentration dependence of the rate constant for reduction was obtained, indicating complex formation preceding electron transfer. Estimates of 3 times 10⁷M^?1 s^?1 and 140–180 s^?l were obtained from these data for the second order rate constants for complex formation, and the limiting first order rate constants for electron transfer, respectively. At neutral pH, a biphasic dependence of the rate constant for ferredoxin reduction on the concentration of NaCl or MgCl₂ was observed. This was interpreted in terms of the electrostatic interactions which occur between ferredoxin and the PSI membrane. In addition, magnesium cations appear to play a specific role in the interaction between PSI and ferredoxin. Thus, the addition of these ions under optimal conditions induces a 6-f-old increase in the electron transfer reaction rate constant, compared with a 2-f-old increase in the presence of an optimal amount of NaCI. This cannot be explained as arising from ionic strength effects. To our knowledge, this is the first time that a direct measurement of the rate constant for the reduction of ferredoxin by the PSI complex has been reported.