COMPARISON OF PHOTOTRAP COMPLEXES FROM CHROMATOPHORES OF RHODOSPIRILLUM RUBRUM, RHODOPSEUDOMONAS SPHEROIDES, AND THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— After dissolution of the membrane structure of chromatophores from Rhodospirillum rubrum, Rhodopseudomonas spheroides , and the R-26 mutant of Rhodopseudomonas spheroides , active phototrap complexes from each have been purified by a column electrophoresis procedure. Phospholipids and transition metals were well separated from the phototrap complex in all three systems. The purified R. rubrum phototrap complex retained a full complement of antenna bacteriochlorophyll and carotenoid pigments which had nearly the same absorbance spectra as in the intact cell, and which delivered absorbed light energy to the phototrap with just as high efficiency as in the intact cell. Sodium dodecyl sulfate (SDS) disc gel electrophoresis using Tris buffer showed that these preparations often contained only two prominent polypeptides of 30,000 ± 2000 and 12,000 ± 4000 mol. wt., and a lesser amount of a third polypeptide of 21,000 ± 2000 mol. wt.
The phototrap complexes prepared from the wild type and the R-26 mutant of R. spheroides were similar, in that a partial separation from antenna pigments occurred during column electrophoresis. Both complexes had prominent polypeptides of 24,000 ± 2000 and 21,000 ± 2000 mol. wt., but no polypeptide of 30,000 mol. wt remained after electrophoresis. A third major polypeptide occurred with a mol. wt. of about 12,000 but seemed identifiable with an incompletely separated antenna pigment fraction. The phototrap complex prepared from the R-26 mutant had a typical reaction center spectrum.
In the case of wild type R. spheroides purification, two distinct protein-pigment complexes separated. Although the absorbance of the bacteriochlorophyll and carotenoid pigments were little changed from those of the in vivo system, different polypeptides in the two fractions were observed by SDS disc gel electrophoresis; only one fraction seemed to be intimately related with the phototrap complex.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE INTERACTION OF PHENAZINE METHOSULFATE WITH REACTION-CENTER PREPARATIONS FROM THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have shown, by means of a kinetic analysis, that phenazine methosulfate (PMS) interacts with reaction-center preparations from the blue-green mutant R26 of Rhodopseudomonas spheroides. At intermediate concentrations of PMS, biphasic decay kinetics of the P870⁺ ESR signal are observed demonstrating that the PMS radical interacts with reaction centers by a specific binding mechanism. With PMS bound to reaction centers, the P870⁺ ESR signal decays in ˜ 1 ms; whereas, in unbound reaction centers the decay is ˜ 120 ms. A model is proposed involving the interaction of PMS on the donor side of P870.     

ISOLATION OF PHOTOCHEMICAL REACTION CENTERS FROM A CAROTENOIDLESS MUTANT OF RHODOSPIRILLUM RUBRUM*

Lauryl dimethyl amine oxide was used to isolate reaction centers from a carotenoid-less mutant, strain G-9, of Rhodospirillum rubrum. These reaction centers have absorption spectra and light or chemically induced difference spectra very similar to those obtained from Rhodopseudomonas spheroides, strain R-26. But, unlike those from Rps. spheroides, they are more labile to higher detergent concentrations and to ammonium sulfate. The cytochrome content was estimated to be less than one per 10 P870.     

LIGHT-DEPENDENT AND LIGHT-INDEPENDENT PRODUCTION OF HYDROGEN GAS BY PHOTOSYNTHESIZING RHODOSPIRILLUM RUBRUM MUTANT C

Abstract. Rhodospirillum rubrum mutant C grew photosynthetically in the light and produced copious amounts of H₂. During light-growth mutant C produced 7.9mmol of H₂ in medium with 9mmol of Na-pyruvate per mg protein. When parent strain R. rubrum S ₁ was grown similarly, these cells only produced a trace amount of H₂. Light-grown mutant C evolved H₂ by H₂-nitrogenase and formic hydrogenlyase. Although both hydrogenases were previously detected in R. rubrum S₁, the activities of the reactions in light-grown mutant C were higher and they operated under different conditions. In the parent strain S₁, the production of nitrogenase was strongly repressed during growth in medium enriched with organic nitrogen and the cells only reduced 0.06 pmol of acetylene per mg protein after 30 min in the light. Under similar conditions, nitrogenase activity measured by the acetylene reduction test in mutant C was 10-fold greater. In addition to nitrogenase, mutant C also produced large amounts of H₂ with formate as an intermediate when the cells were grown with Na-pyruvate. Formic hydrogenlyase in mutant C operated equally well in anaerobic light or dark conditions. The analogous formate oxidation reaction in parent strain S₁ only functioned in the dark. These data, compared with results with R. rubrum S₁ suggested that C was a regulatory mutant. Additional observations suggested that formic hydrogenlyase occurred constitutively in R. rubrum . Pyruvate formate-lyase, however, which produced formate for formic hydrogenlyase, was only detected in the cells after growth in media with Na-pyruvate. The reaction was not formed when R. rubrum was grown in the light in media with dl-malate as the sole carbon substrate.     

ABSORPTION CHANGES IN BACTERIAL CHROMATOPHORES-II. A NEW CHLOROPHYLL-LIKE PIGMENT FROM THE OXIDATION OF CHROMATQPHORES FROM RHODOSPIRILLUM RUBRUM

Abstract— Evidence is presented which points to (at least) two bound forms of bacteriochlor-ophyll present in chromatophores of Rhodospirillum rubrum , both of them readily converted to unbound bacteriochlorophyll (abs. max 770 mμ) when the chromatophores are extracted with acetone or ethanol. Controlled oxidation of the chromatophores with Ir(IV) or with Zn (II) and ferricyanide preferentially destroys the more strongly absorbing pigment (abs. max 880 mμ) but brings about only a slight decrease in the magnitude of the photoinduced absorption changes at 810 and 792 mμ. Such oxidations yield a new pigment, absorbing at 715 mμ in the aqueous preparation and, more strongly, at 680–684 mu when the pigment is extracted into organic solvents. This pigment is formed irreversibly and is therefore different from the material formed by photooxidation of chromatophores. Its visible spectrum and the spectrum of the material formed from it by acidification suggest that it is a chlorophyll-like substance, possibly derived from bacteriochlorophyll by (two-electron) oxidation of one of the dihydropyrrole rings to a pyrrole ring. Directions are given for separation of this pigment from other colored compounds present in the oxidation mixtures.     

FERRIC ION AS PHOTORECEPTOR OF PHOTO- PHOBOTAXIS IN NON-PIGMENTED RHODOSPIRILLUM RUBRUM

Abstract— Rhodospirillum rubrum cultivated aerobically and containing no detectable bacteriochloro-phyll assembled in their phobotactic response into a spot of blue and near UV light. The shape of the action spectrum for the phenomenon was similar to that of the absorption spectrum of ferric ion in aqueous solution. The photophobotactic response was not observed in media containing no ferric ion. Increasing concentration of the ion in the media resulted in an increasing degree of their photophobotactic response. Ferrous ion could not be substituted for ferric ion unless its autooxidation in medium was allowed.
In addition to ferric ion, existence of appropriate anion, by which photoreduction of ferric ion took place efficiently, was necessary to induce an appreciable photophobotactic response. This suggested that the apparent photophobotaxis was, strictly speaking, chemophobotaxis induced by a photochemical product of ferric complex in medium.     

COMPLEX CHARGE RECOMBINATION KINETICS OF THE PHOTOTRAP IN RHODOSPIRILLUM RUBRUM*

Abstract— The dark decay kinetics of excited phototraps in chromatophores prepared from Rhodospirillum rubrum have been quantitatively measured under two different conditions where a direct return of the electron(s) from ubiquinone to the primary donor bacteriochlorophyll cation radical is expected. One of these experimental conditions was with a frozen sample (whole cells, chromatophores, and extracted chromatophores) at 77 K, while the other was a room temperature measurement of a sample of chromatophores from which the loosely bound ubiquinone was first removed by extraction with organic solvents. Results with the latter system were also compared to those obtained with chromatophores plus 1 mM o-phenanthroline. In all cases the kinetics of charge recombination were not pseudo-first order but showed a biphasic pattern. The data at low temperature could be exactly fit by assuming that two major pseudo-first order processes were occurring with half times of 6.5 and 19 ms, 22% decaying by the faster process and 78% by the slower process. Both types of experiments at room temperature could also be fit by assuming two major pseudo-first order processes were occurring, but with somewhat slower half times, 22 and 59 ms. In this case, 20% decayed by the faster process and 80% by the slower process. An equation was developed to fit the data assuming that one ubiquinone molecule was the electron acceptor for two donor units (the duplex model), the dark decay thus taking place in two sequential steps with 50% of the oxidized donor units becoming reduced in each step. The experimental data could be exactly fit using this equation and the above parameters. The data are viewed as best supporting the duplex model for explaining primary photochemical events in these bacteria.     

KINETICS AND MECHANISM OF PHOTO-INDUCED METHYL VIOLOGEN REDUCTION WITH ZINC-MESO-TETRAPHENYLPORPHYRINTRISULFONIC ACID AND HYDROGEN EVOLUTION FROM WATER HYDROGENASE

Abstract— The formation of the cation radical of methyl viologen was established when an aqueous solution containing zinc-meso-tetraphenylporphyrintrisulfonic acid, methyl viologen and mercaptoethanol was irradiated by visible light. The reduction of methyl viologen has been studied kinetically and the reduction rate expressed as follows

where [Zn]₀ is the total amount of zinc compounds in the system.
On the basis of the rate expression, the reaction mechanism is discussed.
On the addition of hydrogenase to the above photoirradiation system, hydrogen evolution was observed by both the irradiation of sunlight and a tungsten lamp.     

A COMPARISON OF DECAY KINETICS OF PHOTO-PRODUCED ABSORBANCE,EPR, AND LUMINESCENCE CHANGES IN CHROMATOPHORES OF RHODOSPIRRILLUM RUBRUM*

–Quantitative comparison of the decay rate for absorbance photochanges in chromatophores of R. rubrum at the major wavelengths of peaks and troughs (280, 365, 385, 433, 605, 763, 790, 810, 850, 865, 890 nm) reveal no major differences under a variety of sample conditions. In addition, the decay kinetics of EPR phtochange at the two environmental potentials used in this study are identical with the absorbance ph;otochange decay. Decay curves for fresh chromatophores, aged chromatophores, and fresh chromatophores at high ionic strength display a variety of half times and curve shapes. All of these data however, may be fit (within 10 per cent) by the equation x=αe^8.2t+βe^0.18t by merely varying the values of α and β. This is interpreted as meaning that any single trapping site may exist in one of two major forms each of which decays following a first order or pseudo-first order reaction. Although the change in decay pattern upon aging is not reversible, that due to high ionic strength is. The time dependencies for two light emission phenomena have been measured as well as their response to the oxidation state of molecules at the trapping site. A major component of long-lived luminescence follows pseudo-second order kinetics with a half time of 38 msec. A very long lived luminescence shows an increse with time which is nearly proportional to the decay of absorbance photochanges.     

菁染料与溴化银相互作用性质的研究

本文用计时电位法及电位滴定研究了十六种不同染料与溴化银之间的相互作用,进一步证明了具有离域π-电子的菁染料才能与卤化银形成络合物的论点。从得到的平衡常数K表明,固体表面上的卤化银-染料与溶液中银离子-染料具有相同键性质,都是银离子与染料离域π-电子作用的结果。     

稀土与染料的相互作用

本文用可见吸收光谱、核磁共振谱、红外光谱等分析方法,研究了Nd、Eu与阳离子染料、酸性染料、活性染料及直接染料之间的相互作用。结果表明稀土与大多数染料之间没有形成稳定的配合物,在中性或酸性条件下与含磺酸基的染料之间只形成磺酸盐,与含羧酸基的染料之间形成羧酸盐。只有与酸性媒介染料之间形成稳定的配合物,其结构与铬及其配合物相似。     

INFLUENCE OF LIGHT ON THE EPR-DETECTABLE, ELECTRON TRANSPORT COMPONENTS IN WHOLE CELL RHODOSPIRILLUM RUBRUM

Abstract —The influence of light upon the electron paramagnetic resonance (EPR)-active components of R. rubrum , both in whole cells and in subcellular particle preparations, is presented. In the whole cells, 16 EPR signals can be detected, 11 of which are influenced by illumination of the bacteria. Four of these are also affected by illumination at 77 K. The effects of K₃Fe(CN)₆, Na₂S₂O₄ and heating upon the signals observed in the whole cells are also presented. The whole cells contain several photoinfluenced, EPR-active components that are missing in the subcellular particle preparations. The kinetics of most of the signals in the whole cells are complex, whereas in the subcellular particles the kinetics are generally monotonic. For comparison, some of the EPR signals detected in whole-cell Chromatium D are also presented. The signals observed in these two strains of bacteria differ considerably. A discussion of the possible identities of the R. rubrum signals and speculation as to the role of some of them in the photosynthetic electron transport system are also included.     

氯取代硫三碳菁染料的合成及性能

本文合成了七个硫三碳菁.其中四个具有桥环的菁染料。测定了它们的电子吸收光谱,讨论了结构对光稳定性的影响。并发现镍络合物能有效地抑制菁染料的光退色。     

KINETICS OF THE PHOTO-INDUCED EPR SIGNAL IN WHOLE-CELL RHODOSPIRILLUM RUBRUM: EFFECTS OF LIGHT INTENSITY, DARK ADAPTATION, TEMPERATURE, AND MICROWAVE POWER

Abstract— Kinetics of the bacteriochlorophyll (P870⁺) electron paramagnetic resonance signal photo-induced at room temperature in whole-cell Rhodospirillum rubrum exhibit transients which are strongly dependent upon the light-dark history of the cells. This paper reports a study of these kinetics as a function of actinic light intensity, light-dark history, temperature and applied microwave power. The simplest interpretation of the observed complexities in the kinetic curves is that the rate of formation and the rate of decay of P870⁺ are controlled by slow dark reactions of the electron-transport chain, and that the rate-controlling reaction is variable during the transition from a dark-adapted to a steady state in the light. With this interpretation, it is possible to measure or to infer order-of-magnitude estimates of the lifetimes of some of the slow reactions.     

INTERACTION OF ACRIDINE DRUGS WITH DNA AND NUCLEOTIDES

Abstract— At high phosphate-to-drug ratios acridine drugs intercalate between hydrogen bonded DNA base pairs causing significant changes in the physico-chemical properties of DNA. The determination of the nature of the strong (or primary) interaction between acridine drugs and DNA is of great importance for elucidating the mode of the biological action of the drugs. Nanosecond measurements have revealed a fast depolarization of the fluorescence of proflavine, one of the most extensively studied acridines, bound to DNA. The electronic structure of the complex, however, is not substantially altered during the lifetime of the excited singlet electronic state of the drug. Guanine has been shown to be responsible for the quenching of the proflavine fluorescence upon binding to DNA. A temperature-jump relaxation study has demonstrated a rather external complexation of this drug with the G-C base pairs; this complex, whose formation occurs in the strong binding region, is distinct from the weak electrostatic complex. The findings that the binding ability of a series of acridines correlates with their basicity and that the drug–binding behavior of methylated DNA is significantly different from that of DNA suggested that specific forces may be also involved in the drug–DNA binding in addition to hydrophobic forces. Recent experiments employing molecular complexes of acridines with nucleotides as model systems have provided strong support for the specificity of the drug-DNA interaction. Hydrogen bonding between the drug and reactive groups of the DNA bases that do not contribute directly to the stability of the helix may be involved in that interaction. The stoichiometry of the proflavine-guanosine 5′-phosphate complex is 1:1. Its association constant increases 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. In view of the proposed importance of the drug–base interaction in explaining the mutagenic properties of acridine drugs and, in particular, of the proposed involvement of the G-C base pairs, this finding emphasizes the possible importance of drug photoexcitation in acridine mutagenesis; it also contributes to the elucidation of photodynamic action. X-ray diffraction studies have recently provided very interesting demonstrations of strong binding of 9-aminoacridine and of the phenanthridine drug ethidium bromide to adenine-uracil base pairs in the crystalline phase. The ability of photoexcited acridine drugs to inactivate viruses has been recently used for therapeutic purposes. The carcinogenic risk involved, however, is still under investigation.     

INTERACTION OF 2,4,6-TRINITROTOLUENE AND ITS ANALOGUES WITH ALDEHYDES. SYNTHESIS OF BENZOANNELATED HETEROCYCLES FROM THE PRODUCTS OF CONDENSATION

ABSTRACT

2,4,6-Trinitrotoluene (TNT) and its sulfonyl analogue 2-isobutylsulphonyl-4,6-dinitrotoluene undergo smooth condensation with chloral and fluoral to give 2-R-4,6-dinitrophenyl-1-(trihalomethyl)ethanols which easily cyclize to give 4-R-6-nitro-2-trihalomethyl-2,3-dihydrobenzo[b]furans (R=NO₂, i-Bu; halogen = F or Cl) in the presence of K₂CO₃. 2-R′-sulphonyl-4,6-dinitrotoluenes, prepared from TNT, condense with aromatic aldehydes to form 1-(2-R′-sulphonyl-4,6-dinitro)-2-arylethenes in which the ortho-nitro group, upon interaction with NaN₃ was selectively substituted by the azido group. Thermolysis of the obtained azides gave 2-aryl-4-R′-sulphonyl-6-nitroindoles (R′ = Ph, i-Bu, PhCH₂). Such N-methylated indole (R′ = i-Bu) was regioselectively aminated.     

INACTIVATION AND MUTAGENESIS OF Herpes VIRUS BY PHOTODYNAMIC TREATMENT WITH THERAPEUTIC DYES

Dyes which photosensitize membranes may be clinically useful for photodynamic treatment (PDT) of Herpes simplex virus (HSV) infections. It is important to determine whether the enveloped HSV can be inactivated via membrane damage without affecting the genetic material. Selection of appropriate PDT conditions, including the choice of dye, could minimize viral mutagenesis. We determined the mutagenesis caused by PDT employing three membrane-photosensitizing dyes of potential use in cancer photochemotherapy (Photofrin II, polyhematoporphyrin esters, zinc phthalocyanine tetrasulfonates) and a DNA-photosensitizing dye (proflavine sulfate). The effects were compared to those caused by exposure of HSV to ultraviolet radiation (UV). The procedure consisted of incubating HSV with microgram/ml (microM) concentrations of the dye, irradiating the samples with broad spectrum visible/near-UV radiation (Daylight fluorescent lamps) and assaying the survival of the treated HSV. Zinc phthalocyanine was the most potent dye per absorbed photon for inactivating HSV. In parallel with determination of survival, progeny of the surviving virus were grown for determination of mutagenesis. The progeny virus was harvested and subsequently assayed in the presence and absence of 40 micrograms/ml iododeoxycytidine (ICrd) to determine the frequency of mutation to ICrd resistance. Mutation frequencies were determined for progeny from the 1-4% survival level. For PDT with each membrane-photosensitizing dye, only zinc phthalocyanine increased the mutation frequency over the untreated control. This increase was less than 2-fold. Proflavine increased the mutation frequency 2-3 fold over the untreated control. Ultraviolet produced a 15-20 fold increase over the untreated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

EFFECTS OF 2,5-DIBROMO-3-METHYL-6-ISOPROPYL BENZOQUINONE (DBMIB) ON PHOTOCHEMICAL EVENTS IN RHODOSPIRILLUM RUBRUM*

Abstract. The quinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl benzoquinone, DBMIB)? was used to inhibit early photochemical changes in chromatophores of the photo-synthetic bacterium, Rhodospirillum rubrum. With continuous illumination with near infrared light, we observed an approximately threefold decrease in efficiency of P865⁺ formation at 100–200 μM DBMIB as measured by absorbance changes at 605 or 430 nm. However, with continuous illumination of several seconds duration, maximal absorbance changes were observed. At low concentrations (1–10 μM) of DBMIB, a decrease in the decay rate of the ΔA₄₃₀ or ΔA₆₀₅ was observed. Using a short (2–10 μs), low intensity light pulse for excitation, we observed that DBMIB inhibits P865⁺ formation. The concentration dependency of this inhibition corresponds to that seen with continuous illumination. We tested the possibility that DBMIB was displacing the first stable electron acceptor, which has been shown to be a tightly bound ubiquinone (UQ) in R. rubrum. Chromatophores prepared from cells grown with ¹⁴C-p-hydroxybenzoic acid in their media provided a sample in which the benzoquinones were labelled specifically with ¹⁴C. These labelled chromatophores were first extracted with petroleum ether to remove the loosely bound ubiquinone pool and then were treated with DBMIB. DBMIB displaced about 1/2 of the remaining tightly-bound UQ present. The DBMIB inhibition was tested for reversibility by adding an excess of UQ to DBMIB-treated samples. In these cases, restoration to 80% could be achieved from samples which were only 20% active relative to the untreated samples. A similar inhibitory effect by DBMIB was also demonstrated in whole cells of Rhodospirillum rubrum, the G-9 mutant of Rhodospirillum rubrum, Rhodopseudomoms sphaeroides, Rhodopseudomonas capsulata, and Chromatium vinosum. The mechanism for inhibition is most consistent with displacement of, or interference with the first stable electron acceptor (ubiquinone) rather than by random quenching of excited states at the level of antenna bacteriochlorophyll.