A HIGHLY REACTIVE HETEROATOM ANALOG OF RETINAL AND ITS INTERACTION WITH BACTERIORHODOPSIN

Abstract— C₁₈ formate ester (5) [2-(6-methyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3E,5E,7E-octatrienyl formate], a highly reactive analog of retinal, was synthesized and its interaction with bacterioopsin studied. The formate ester, in the absence of purple or bleached membrane, undergoes very rapid reaction (t_l/2= 0.9 min) in neutral buffer but with membrane present it diffuses more rapidly into the membrane where it reacts slowly. Incorporation of 5 in the membrane results in a 38 nm (3900 cm^-1) red shift which remains after reconstitution with retinal. Similar experiments with the corresponding C₁₈ alcohol (4) results in a red shift, but this absorption blue shifts upon reconstitution with retinal. Washing the formate ester-treated membrane with bovine serum albumin or the corresponding lyophilized preparation with hexane, treatments that remove retinal oxime, fails to remove the UV-visible absorption, suggesting that a covalent bond between the C₁₈ moiety and a nucleophilic group of the protein has probably formed.     

A HIGHLY REACTIVE HETEROATOM ANALOG OF RETINAL AND ITS INTERACTION WITH BACTERIORHODOPSIN

Abstract
C₁₈ formate ester (5) [2-(6-methyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3E,5E,7E-octatrienyl formate], a highly reactive analog of retinal, was synthesized and its interaction with bacterioopsin studied. The formate ester, in the absence of purple or bleached membrane, undergoes very rapid reaction (t_l/2= 0.9 min) in neutral buffer but with membrane present it diffuses more rapidly into the membrane where it reacts slowly. Incorporation of 5 in the membrane results in a 38 nm (3900 cm^-1) red shift which remains after reconstitution with retinal. Similar experiments with the corresponding C₁₈ alcohol (4) results in a red shift, but this absorption blue shifts upon reconstitution with retinal. Washing the formate ester-treated membrane with bovine serum albumin or the corresponding lyophilized preparation with hexane, treatments that remove retinal oxime, fails to remove the UV-visible absorption, suggesting that a covalent bond between the C₁₈ moiety and a nucleophilic group of the protein has probably formed.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

A Resonance Raman Study Of the C=C Stretch Modes in Bovine and Octopus Visual Pigments with Isotopically Labeled Retinal Chromophores

Abstract— Previous resonance Raman spectroscopic studies of bovine and octopus rhodopsin and bathorhodopsin in the C–C stretch fingerprint region have shown drastically different spectral patterns, which suggest different chromophore-protein interactions. We have extended our resonance Raman studies of bovine and octopus pigments to the C=C stretch region in order to reveal a more detailed picture about the difference in retinal-protein interactions between these two pigments. The C=C stretch motions of the protonated retinal Schiff base are strongly coupled to form highly delocalized ethylenic modes located in the 1500 to 1650 cm⁻¹ spectral region. In order to decouple these vibrations, a series of 11,12-D₂-labeled retinals, with additional 13C labeling at C₈, C₁₀, C₁₁ and C₁₄, respectively, are used to determine the difference of specific C=C stretch modes between bovine and octopus pigments. Our results show that the C₉=C₁₀ and C₁₃=C₁₄ stretch mode are about 20 cm⁻¹ lower in the Raman spectrum of octopus bathorhodopsin than in bovine bathorhodopsin, while the other C=C stretch modes in these two bathorhodopsins are similar. In contrast, only the C₉=C₁₀ stretch mode in octopus rhodopsin is about 10 cm⁻¹ lower than in bovine rhodopsin, while other C=C stretches are similar.     

AN ULTRASENSITIVE BIOASSAY FOR THE DETECTION OF FUROCOUMARINS AND OTHER PHOTOSENSITIZING MOLECULES

Abstract— A photobiological assay based upon inhibition of growth in the DNA repair-deficient bacterium E. coli B _s-1, is described for the analysis of a number of photosensitizing agents. The lower limits of detection were as follows: psoralen 5 × 10^-11g; 5-methoxypsoralen 1 × 10^-9 g; 8-methoxypsoralen 1 × 10^-9 g; 4,5',8-trimethylpsoralen 1 × 10-¹¹ g; angelicin 5 × 10^-9 g; 5,7-di-methoxycoumarin 1 × 10^-7 g; isoimperatorin 5 × 10^-9 g; dictamnine 1 × 10^-8 g; oxypeucedanin 5 × 10^-7 g; 5-nitroxanthotoxin 5 × 10^-7 g; and α-terthienyl 1 × 10^-6 g. All active compounds with the exception of α-terthienyl were more easily detected by several orders of magnitude by E. coli B _s-1 than with the normal wild type E. coli. 5—Geranoxypsoralen and isopimpinellin were not active. The application of this technique, after TLC, to the analysis of complex mixtures from lemon oil, oil of bergamot, Heracleum lanatum, Angelica dawsonii , and celery and parsnip is illustrated. The bioassay described is more rapid and sensitive than previously published methods, permits replica plates to be made, and allows tentative identification of the photosensitized molecular target.     

MECHANISMS OF PHOTOELIMINATION REACTIONS OF ALKYL BENZOHYDROXAMATES

Abstract— Photolysis at 254 nm of alkyl benzohydroxamates [C, H, CONHOR: R = CH₃ H₂CH₃ CH(CH₃)₂, CH₂C₆H₅ CH(CH₃)C₂H₅ CH(CH₃)- n -C₆H₁₃] in acetonitrile or hydrocarbon solvents gives benzamide. These reactions can be sensitized by benzophenone (at ca. 350 nm) and are quenched by cis-piperylene. Racemization occurred when 2-octyl (+)-benzohydroxamate was irradiated in cyclohexane. These results are consistent with a mechanism involving a triplet biradical. Photolysis of phenyl benzohydroxamate [C₆H₅CONHOC₆H₅] and benzyl N -methylbenzohydroxamate [C₆H₅CON-(CH₃)OCH₂Q₆H₅] cannot be quenched with ris-piperylene and appear to be singlet reactions.     

Photocycloaddition Reactions of Pyrazinopsoralen with Simple Olefins

Direct photolyses of pyrazinopsoralen (PzPs) with excess olefins such as dimethyl fumarate (DMFu), dimethyl maleate (DMMa) and dimethyl ethylidenemalonate (DMEM) gave C₄-photocycloadducts. The photoproducts were determined to be 1:1 C₄-cycloadducts formed through the addition of 4',5'-furan double bond of the excited P_zP_s to the olefins. The fluorescence of P_zP_s was quenched by olefins with rate constants on the order of 10₉–10₁₀ M -¹/s. The appearance of the long-lived fluorescence component implies a singlet exciplex mechanism for the photocycloaddition reaction of PzPs with excess olefins.     

ULTRAVIOLET RADIATION INHIBITS BOTH ACCESSORY CELL AND RESPONDER T CELL FUNCTION IN THE PROLIFERATIVE RESPONSE TO HAPTENATED-SELF

Abstract— Photoprotection i. e. the increased resistance of the cells preilluminated with near ultraviolet light (300–380 nm) to the lethal action of 254 nm radiations is observed in wild-type Escherichia coli B cells (which exhibits the Fil phenotype) but requires either an integrated prophage or a rec A mutation to be detected in E. coli K12 strains. Here we have demonstrated that significant photoprotection occurs in an E. coli K12 rec A⁺ cell containing the Ion allele which is responsible for filamentous growth (Fil phenotype) after 254 nm irradiation. The Fil phenotype can be suppressed by the sfi A of sfi B suppressor genes. Since the E. coli K12 rec A⁺ Ion sfi B strain exhibits no more photoprotection, these data support the conclusion that in Ion strains photoprotection is due to the abolition of the 254 nm induced filamentation by the near ultraviolet treatment. In addition, we show here that near ultraviolet illumination of the cells leads to a severe restriction of the bulk protein synthesis, as well as of the inducibility of β-galactosidase and tryptophanase. These effects are observed only in nuv ⁺ cells that contains 4-thiouridine the chromophore responsible for photoprotection. We propose that in Ion (lysogenic) strains, photoprotection is due to prevention of the SOS response. During the growth lag, the low residual level of protein synthesis does not allow the induction of the SOS response and accordingly prevents filamentation (the lytic cycle). Concomitantly the SOS triggering signals are eliminated via DNA repair.     

WEIGLE REACTIVATION IN ACINETOBACTER CALCOACETICUS

Abstract— Weigle (W)-reactivation was demonstrated in Acinetobacter calcoaceticus for the UV-irra-diated lysogenic phage P78. The reactivation factor (survival of irradiated phage on irradiated bacteria/ survival on unirradiated bacteria) reached a maximum value of 20. This was obtained at UV-doses giving phage and host survivals of about 5 times 10_-6 and 1 times 10_-1, respectively. Intracellular development of W-reactivated P78 was followed by one-step growth experiments. Conditions which allowed maximal W-reactivation also extended the period of phage production and yielded a somewhat reduced burst size.     

THE EFFECT OF ALIPHATIC ALCOHOLS ON FLUORESCENCE QUENCHING AND FLUORESCENCE POLARIZATION OF LUMINESCENCE PROBES IN HEXADECYLTRIMETHYLAMMONIUM BROMIDE MICELLES

Abstract— The fluorescence quenching of the indole chromophore by NO⁻₂ and the fluorescence depolarization of several luminescence probes in aqueous solutions containing hexadecyltrimethylammonium bromide (HDTBr) were measured as a function of added C₂–C₄ aliphatic alcohol concentration. The fluorescence decay profiles of pyrene in the micellar solutions were also measured to estimate the aggregation number of the micelles. The addition of n -butyl alcohol significantly reduces the fluorescence quenching rate and the aggregation number and increases the extent of fluorescence depolarization in HDTBr micellar systems. The addition of ethyl alcohol shows a similar but smaller effect.     

THE PRESENCE OF PHOTOREACTIVE β-CARBOLINE FLUOROPHORES IN WEATHERED WOOL KERATIN

Abstract Solvent-cleaned Merino wool, which was enzymatically digested with protease K, displayed a fluorescence maximum at 500 nm when excited at 430 nm. The yield of this emission was approximately 15 times greater for a fiber tips digest than for a digest of the mid-length region of the same fibers.
Separation of the components in the wool tip digest by silica gel thin-layer chromatography revealed the presence of several fluorescent species. The chromatographic mobility of these species was similar to the behavior observed in a preparation of authentic 1-methyltetrahydro-P-carboline, 1,3-dicarboxylic acid.
Mass spectroscopy of the fluorescent compounds in the wool tip digest displayed molecular ions, [M + H]⁺ with m/z = 273.0890 and 257.0560, and molecular weights that define the formulae C₁₄H₁₂N₂O₄ and C₁₁H₈O₄N₂, respectively, which correspond to β-carboline 1,3-dicarboxylic acids. Mass spectral evidence also indicates the presence of two other β-carbolines.     

Physicochemical Studies of Demetalation of Light-harvesting Bacteriochlorophyll Isomers Purified from Green Sulfur Photosynthetic Bacteria

Demetalation kinetics of bacteriochlorophylls (BChls) c, d and e from green sulfur photosynthetic bacteria were studied under weakly acidic conditions. Demetalation rate constants of BChl e possessing a formyl group at the 7-position were significantly smaller than those of BChls c and d , which had a methyl group at this position. The activation energy of demetalation of 3¹ R -8,12-diethyl([E,E])-BChl e was 1.5-times larger than that of 3¹ R -[E,E]-BChl c . ¹⁵N-labeled 3¹ R -[E,E]-BChls c and e were purified from cells of green sulfur bacteria grown in a medium containing ¹⁵NH₄Cl, and their ¹⁵N NMR spectra were measured. The chemical shifts of N₂₁, N₂₂ and N₂₃ atoms of 3¹ R -[E,E]-BChl e were lower-field shifted than those of 3¹ R -[E,E]-BChl c , respectively, and especially the difference in chemical shifts of N₂₂ was significantly large. These results suggest that the electron-withdrawing formyl group at the 7-position of BChl e affected an electronic state of the chlorin macrocycle and caused BChl e to be more tolerant for removal of the central magnesium compared with BChls c and d .     

Fluorescent Dimers of Merocyanine 540 (MC540) in the Gel Phase of Phosphatidylcholine Liposomes

Abstract— Fluorescence emission from merocyanine 540 (MC540) dimers was observed in dipalmitoylphosphatidylcholine (DPPC) vesicles. This unusual behavior was observed only for vesicles in the gel-phase state. No dimer fluorescence was observed either in monopalmitoylphosphati-dylcholine (C₁₆PC) micelles or in liquid-crystalline DPPC vesicles, indicating that dimer fluorescence efficiency increases in highly packed interfaces. The excitonic theory of Kasha was used to interpret the spectral features. The overall fluorescence quantum yield (φ_r) decreases with decreasing lipid: probe ratio, not only because of the presence of a weakly fluorescent dimer that absorbs a high fraction of the total absorbed light but also due to quenching of monomer emission. This suggests the existence of probe domains. The dimer fluorescence quantum yields (φ_m) were estimated in DPPC large unilamellar vesicles (LUV) and DPPC multilamellar vesicles. The dependence of φ_r with probe concentration is compatible with values of φ_m lower than 0.05. The dimerization equilibrium of MC540 in C₁₆PC micelles and DPPC-LUV was also studied. Apparent dimerization equilibrium constants, K_dapp and dimer absorption spectrum were calculated in C₁₆PC micelles for the first time. The dimerization equilibrium constant in DPPC-LUV was calculated and discussed in terms of the fraction of volume occupied by the lipid phase.     

STRUCTURE-ACTIVITY RELATIONSHIP OF PORPHINES FOR PHOTOINACTIVATION OF BACTERIA

Abstract— The antibacterial photodynamic effects of uncharged ( o -tetrahydroxyphenyl porphine [THPP], m -THPP and p -THPP), cationic (5,10,15,20-tetra[4- N -methylpyridyllporphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS₄]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS₄ showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS₄, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.     

NMR ANALYSES OF TRYPTOPHAN-8-METHOXYPSORALEN PHOTOREACTION PRODUCTS FORMED IN THE PRESENCE OF OXYGEN

Proton-made spectroscopy was performed on solutions of l -tryptophan and 8-mcthoxypsoralen (8-MOP) in either D₂O or DMSO-d₆ in the presence of oxygen before and after irradiation with 360 nm monochromatic light. Irradiation results in the loss of hydrogen atoms at the 3. 4 and 4'. 5' positions of 8-MOP and at the indole C₂ position of tryptophan. Changes in the aliphatic regions of the spectra also occur with irradiation. It is postulated that generation of photoreaction products between 8-MOP and tryptophan involves the 3.4 and 4'.5' positions of 8-MOP and the imidazole moiety of tryptophan.
Reprint requests to: Dr J. Megaw, Laboratory for Ophthalmic Research, Emory University School of Medicine, Atlanta. Georgia 30322. USA.     

PREPARATION AND CHARACTERIZATION OF THYMIDINE-8-METHOXYPSORALEN-TETRAMETHYLETHYLENE PHOTODIADDUCTS

Abstract A 8-methoxypsoralen<₄⁵₅⁶>thymidine C₄-monoadduct was photolyzed with tetramethylethylene to obtain an analogue of thymidine<₅⁶₄⁵>-methoxypsoralen<₃⁴₅⁶>thymidine C₄-diadducts. The structure of the adduct was determined by physical methods and a triplet manifold was proposed as the reactive state for the photocycloaddition reaction.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–III. SPIN TRAPPING OF PHOTOLYSIS PRODUCTS FROM SULFANILAMIDE ANALOGS

The photodecomposition of sulfanilamide (4-aminobenzenesulfonamide), sulfacetamide. sulfathiazole. sulfadiazine, carbutamide and tolbutamide has been studied using the spin traps 2-methyl-2-nitrosopropanc and 5,5-dimethyl-l-pyrroline-l-oxide. The following radicals were trapped during the photolysis of sulfanilamide in aqueous solution: H' and HNC₆H₄SO₂NH, (α-fission). SO₂NH₂ and C₆H₄NH₂ (δ fission). H₂NC₆H₄SO₂ and NH₂ (δ-fission). Although the C.,H₄SO₂NH₂ and the SO; radicals were also detected these were not formed directly by homolytic bond fission. Homolytic bond fission was also observed during the irradiation of sulfacetamide (α.δ), sulfadiazine (α). carbutamide (α,δ) and tolbutamide (δ). All of the analogs, with the exception of tolbutamide, generated the SO; radical. Sulfacetamide, sulfadiazine and carbutamide generated the C₆H₄SO_2;NHR radical by some process that did not involve homolytic bond fission. The free radicals generated by these agents may play an important role in their phototoxic and photoallergic effects.     

PATTERNS OF NUCLEIC ACID AND PROTEIN SYNTHESIS DURING MODIFICATION OF LETHAL ULTRAVIOLET LESION

Abstract— We have shown previously that the viability of E. coli B.U.^- and E. coli B. try^-could be restored after u.v.-irradiation if post-irradiation incubation took place in the presence of chloramphenicol or in the absence of an essential metabolite. By way of contrast, the viability of the u.v.-irradiated E. coli K.₁₂ meth^- decreased rapidly during post-irradiation incubation in the presence of chloramphenicol or in the absence of methionine. During the study of the biosynthesis of nucleic acids and protein in the above strains we made the following observations.
Protein synthesis was permanently inhibited in both amino acid requiring strains during post-irradiation incubation in the absence of the respective essential metabolite. In E. coli B.U.^- a slow synthesis of protein could be observed even if post-irradiation incubation took place in the absence of uracil.
DNA synthesis was completely and permanently inhibited in both E. coli B. try^- and E. coli K.₁₂ meth^- during post-irradiation incubation in the absence of the respective essential amino acid. In E. coli B.U.^-, however, DNA synthesis resumed after a prolonged lag even if the bacteria were incubated after u.v.-irradiation in the absence of uracil.
RNA synthesis was completely and permanently inhibited in both E. coli B. mutants, the uracil requiring and the tryptophan requiring, if they were incubated after irradiation in the absence of uracil and tryptophan respectively. In E. coli K.₁₂ meth^- a small amount of RNA is synthesized during post-irradiation incubation in the absence of the essential amino acid.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

THE EFFECT OF 1,4-DIAZABICYCLO[2.2.2]OCTANE ON THE RADIOSENSITIVITY OF BACTERIA

Abstract— Hydroxyl radicals ('OH) are scavenged by 1,4-diazabicyclo[2.2.2]octane (DABCO) at a diffusion-controlled rate of 1.25 ± 0.1 × 10⁹ M ^-1s^-1. Unlike other efficient 'OH scavengers which exhibit protection of bacteria against irradiation both in oxic and hypoxic conditions, DABCO has been shown to protect Serratia marcescens and various strains of Escherichia coli only in oxic conditions.
DABCO appears to eliminate a component of the sensitization afforded by oxygen in all strains of E. coli tested. The level of this protection increases from ∼15% in the wild type AB 1157 to ∼100% in the recA uvrA mutant AB 2480. It is suggested that DABCO protects against lethal events that can occur on macromolecules other than DNA such as the cell membrane.
Results with added glycerol, as well as work in D₂O solution, indicate that DABCO is more likely to be acting by scavenging radicals rather than by quenching ¹O₂. If ¹O₂ is a component of the sensitization afforded by oxygen, then it is unlikely to be formed in a hydrophilic environment in the cell.