RED LIGHT-INDUCED REDUCTION OF A PARTICLE-ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Abstract— In the presence of methylene blue, red light causes the reduction of a h-type cytochrome in particulate fractions from corn coleoptiles. Two types of difference spectra for the cytochromes in these fractions are presented: (a) red light-minus-dark in the presence of methylene blue, and (b) dithionite-reduced-minus-oxidized. Comparison of these spectra shows that photoexcited methylene blue selectively reduces a b-type cytochrome which constitutes at most only 30% of the total dithionite-reducible cytochrome present in the most active fractions. The photoreducible cytochrome has an alpha band at room temperature near 557 nm. Bleaching of methylene blue precedes cytochrome reduction under appropriate conditions, suggesting that the photoreduced dye is donating an electron to the cytochrome. This electron transfer does not involve a flavin, at least as judged by the absence of light-induced spectral changes attributable to flavins. Preliminary kinetic studies suggest that EDTA provides the pool of electrons for the reaction. The cytochrome cannot be assigned exclusively either to mitochondria or to endoplasmic reticulum, as judged by its sedimentation properties. These results and the current literature are discussed in the context of the hypothesis that this b-type cytochrome may be involved in the photoreception mechanism for blue and uv light in vivo.     

THE USE OF SPIN LABEL OXIMETRY IN THE STUDY OF PHOTODYNAMIC INACTIVATION OF CHINESE HAMSTER OVARY CELLS

Spin label oximetry has been used to study the effects of photosensitizer hematoporphyrin on oxygen consumption in Chinese hamster ovary cells. These measurements demonstrate that, in the presence of the sensitizer, there occurs (i) an increase in total oxygen consumption during irradiation and (ii) a decrease in oxygen consumption following irradiation. These results are attributed to the effects of photosensitized oxygen consumption and decreased cellular respiration due to cell inactivation. Thus the inhibition of oxygen consumption after irradiation was shown to correlate with decreased cell survival measured in cell culture experiments. Possible mechanisms of inactivation and extensions of the oximetric approach are discussed.     

包覆NIPAM-ODA双亲共聚物的脂质体的温控释放行为

为了研究对温度敏感的双亲性共聚物包覆的脂质体的温控释放行为,合成了N-异丙基丙烯酰胺(NIPAM)和丙烯酸十八酯(ODA)的共聚物。利用荧光探针法研究了共聚物水溶液随温度升高时出现的LCST(lower critical solution temperature)现象,表明该高分子在温度升高到30℃以上时存在着明显的相分离行为。5(6)-羧基荧光素(5(6)-CF)为标记物,研究了高分子包覆的小单层脂质体(small unilamelar vesicles)的释放行为。发现在温度低于30℃时,5(6)-CF的释放百分率比未包覆高分子的脂质体要低;而当温度升高到30℃以上时,其释放百分率明显提高。这种温控释放行为和包覆在脂质体上的高分子在其LCST时存在的相分离行为有关。进一步利用荧光偏振法研究了脂质体膜在包覆高分子后的流动性变化,发现:在温度低于30℃时,其流动性随温度升高而增大;而在温度高于30℃时,脂质体膜流动性随温度升高而降低,进一步证实了高分子在其LCST以上时对脂质体膜的破坏作用。     

AN IDENTIFICATION OF THE RADICAL GIVING RISE TO THE LIGHT-INDUCED ELECTRON SPIN RESONANCE SIGNAL IN PHOTOSYNTHETIC BACTERIA

Abstract— A reaction-center fraction isolated from Rhodopseudomonas spheroides chromatophores exhibits light-induced changes in its optical and electron spin-resonance (ESR) spectra. In particular, a bleaching at 870nm (P870) has been found to be closely correlated with the appearance of an ESR signal with a g factor of 2.0025 and a peak-to-peak line width of 10 G. The ESR signal is indistinguishable from light-induced signals found in chromatophores or whole cells.
A careful measurement of the spin concentration showed that the ratio of the light-induced spins to bleached P870 molecules is 1.1 ± 0.1. In addition the formation and decay kinetics are identical within experimental error under a variety of experimental conditions.
Previous work has shown that P870 is a bacteriochlorophyll molecule in a specialized environment and that the bleaching signifies oxidation. The present work provides strong evidence that the photo-bleaching of P870 produces the radical cation of bacteriochlorophyll, P870⁺, and that this radical is the source of the ESR signal in whole cells.
The quantum yield for the bleaching of P870 in reaction centers has been measured, using actinic light of wavelengths 880, 800, 760 and 680 nm. For light absorbed at 880 or 800 nm the efficiency is close to 100 per cent. In a coupled reaction, the oxidation of mammalian cytochrome c by P870⁺ proceeds with nearly the same efficiency.
The above results place definite limits on the possibilities for the identity of the primary acceptor. These possibilities are discussed.     

CHARACTERIZATION OF RADICAL ADDUCTS FORMED DURING PHOTOCHEMICAL SPIN TRAPPING IN LIPOSOMES

There exists an urgent need to monitor radical reactions in biological membranes. With the exception of a few studies in model and natural membranes, most biological spin trapping reactions have been confined to homogeneous media. In the present work we have devised a methodology by which spin trapping reactions can be investigated in liposomes composed of a fully-saturated phospholipid, dimyristoylphosphatidylcholine. Using photochemical spin trapping, we have detected and characterized the formation and partitioning of spin adducts in liposomes. Benzophenone (a lipid-soluble photosensitizer) and benzoyl peroxide (a lipid-soluble radical initiator) have been used to generate free radicals from hydrogen donors during photolysis in liposomes. Both water-soluble and lipid-soluble spin traps have been used to trap the radicals.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE: A KINETIC STUDY OF ENDOGENOUS LIGHT-INDUCED FREE RADICALS IN REACTION CENTER PREPARATIONS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have detected, by means of a kinetic analysis, a rapidly decaying signal in reaction center preparations from the R26 blue-green mutant of Rhodopseudomonas spheroides. This signal, which we designate Signal B3, is essentially the same as that seen previously in iron-free preparations. Signal B3 decays at 20°C with a 1/ e time of ˜ 3 ms and exhibits an activation energy of 5 ± 1 kcal mol^-1 over the temperature range 0–30°C. Extraction with isooctane completely eliminates Signal B3, whereas readdition of exogenous ubiquinone-30 completely restores the signal. o -Phenanthroline has no effect on Signal B3. We discuss these results in terms of a model in which the primary acceptor is an iron-ubiquinone complex with excess ubiquinone serving as a secondary electron acceptor pool.     

AN ELECTRON SPIN RESONANCE STUDY OF MEROCYANINE 540-MEDIATED TYPE I REACTIONS IN LIPOSOMES

The merocyanine 540 (MC540)-mediated reduction of nitroxide spin labels in a liposomal system was examined using electron spin resonance (ESR) spectroscopy. Spin label reduction was light driven, and occurred in liposomes composed of both fully-saturated (dimyristoyl) and mono-unsaturated (1-palmitoyl-2-oleoyl) phosphatidylcholine. Loss of the nitroxide ESR signal was enhanced by the physiological electron donors glutathione, cysteine, and NADPH; and was strongly inhibited by the presence of molecular oxygen. Nitroxides reduced in the presence of MC540 alone could be regenerated either by purging the sample with air or by the addition of ferricyanide, indicating that the ESR signal loss was due to reduction to the corresponding hydroxylamines. Only partial regeneration was attained for nitroxides reduced in the presence of glutathione, cysteine, or NADPH. Reduction rates for the lipophilic spin labels, 5-, 12-, and 16-doxyl stearic acid, were not influenced by the position of the nitroxide moiety along the alkyl chain, however reduction of spin labels occupying primarily the aqueous phase was much slower. These studies demonstrate that MC540 can initiate oxidation/reduction (Type I) reactions. Such Type I processes may augment the effects of singlet oxygen in MC540-mediated photodynamic therapy.     

SYNTHIESIS OF THIOAMIDES FROM NITRILES AND HYDROGEN SULPHIDE IN THE PRESENCE OF PHASE-TRANSFER CATALYSTS

Abstract

The synthesis of thioamides⁽¹⁾ from nitriles generally involves base catalyzed reaction of nitriles with hydrogen sulphide in a polar organic solvent.     

THIN LIQUID FILMS FROM WATER-BASED DISPERSIONS OF CELLULOSE ACETHOPHTHALATE IN THE PRESENCE OF PILOCARPINE HYDROCHLORIDE

The influence of different surfactants on the possibility to obtain fine dispersions with a comparatively high percentage of cellulose acethophthalate (CAP) has been investigated. A method for obtaining concentrated dispersions of CAP including the anti-glaucoma drug pilocarpine hydrochloride has been proposed.

Thin liquid films formed from aqueous CAP dispersions on a glass surface have been investigated as a model of tear films in the eye. The film thickness has been determined by the microinterferometric method. The kinetics of thinning has been studied and discussed.     

THE PHOTOLYSIS OF TRYPTOPHAN IN THE PRESENCE OF OXYGEN

Abstract— Quantum yields for the destruction of tryptophan by a single 500 J flash in aqueous solution have been determined over the pH range 1–13 in both air-equilibrated and nitrogen-saturated conditions. When these quantum yields are compared with the quantum yields for radical formation and photoejection of electrons, it is found that there is good agreement only for the nitrogen-saturated case. In air-equilibrated solutions of tryptophan, there is a large disparity between the measured degradation quantum yields and those for photoejection of electrons and radical formation. Oxygen, therefore, is playing a major role in the photochemical decomposition and it is proposed that the major reaction which occurs, under normal atmospheric conditions, is the reaction of the lowest triplet excited state of tryptophan with oxygen.
Preliminary photolysis-product distributions against pH are discussed, and indicate that a total of nine major products are formed in the presence of oxygen.     

FLASH-INDUCED FAST CHANGE IN SURFACE POTENTIAL AND FLUIDITY OF PURPLE MEMBRANE STUDIED BY SPIN LABEL METHOD

Abstract— Flash-induced changes in surface potential and fluidity of purple membrane were studied by a spin label method. Changes in surface potential and fluidity were monitored by observing the distribution of charged and uncharged spin labels between the purple membrane and the aqueous phase.
On flash illumination, a transient hyperpolarization of the surface potential and a transient fluidization of the membrane hydrophobic region are induced. The former may probably reflect the proton movement near the purple membrane surface, while the latter may result from photo-induced conformational changes of bacteriorhodopsin.
The two events are different in time course. The relationships between the two events, and the formation and decay of the intermediates of bacteriorhodopsin in the photoreaction cycle are discussed.     

THE ELECTRON SPIN POLARIZATION OF THE DONOR-TRIPLET STATE IN NATIVE and MODIFIED PHOTOSYNTHETIC REACTION CENTERS FROM

The electron spin polarization (ESP) pattern of the donor-triplet state (P^R) of reaction centers (RC's) of the purple bacterium Rhodobacter (formerly Rhodopseudomonas) sphaeroides R-26 was investigated. δm =±1 triplet EPR spectra were recorded of unmodified RC's as well as of RC's from which Fe²⁺ or ubiquinone was removed, or ubiquinone was substituted by menaquinone.
The relative amplitude of the Y peaks in the triplet EPR powder spectrum of P^R decreases when the temperature is increased from 8 K to 100 K in RC's with an intact quinone-iron complex. This decrease is more pronounced when the primary ubiquinone is substituted by menaquinone. These observations provide further support for the hypothesis that the observed lineshape of the P^R triplet state EPR spectrum reflects the presence of a third electron spin, magnetically coupled to I^- in the P⁺I^- radical pair, as suggested by Van Wijk et al. (1986) (Photobiochem. Photobiophys . 11, 95–100). Our observations suggest that this phenomenon may be general in purple bacteria.     

HYDRODYNAMIC EFFECTS IN THE PHOTOSENSITIZED LYSIS OF LIPOSOMES*

Abstract— The photosensitized lysis of phosphatidylcholine liposomes incorporating methylene blue in the membrane or in the presence of external methylene blue is promoted by hydrodynamic agitation concurrent with or subsequent to irradiation with red light. The results implicate the attack of singlet oxygen on an unsaturated lipid component as the key photochemical step, which is followed by additional membrane damage induced by hydrodynamic action.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

LASER MEDIATED RELEASE OF DYE FROM LIPOSOMES

Liposomes made from phospholipids and containing sulforhodamine dye (1-50 mM) have been irradiated with nanosecond and picosecond laser pulses. Individual liposomes were locally heated by laser absorption of dye dimers during a single laser pulse, and heating was sufficient to release the liposome contents. The extent of dye release produced by a single laser pulse was shown to be quantitatively dependent on several interdependent variables, including dye concentration, liposome size, laser excitation parameters and initial temperature of the dye-liposome system. Fluorescence lifetime data having three components have been obtained and analyzed in terms of three dye environments. Quantitative estimates support a photo-induced thermal mechanism for liposome lysis and release of its contents. These results may be useful for laser induced delivery of therapeutic agents or other applications of lasers in biological systems.     

三乙醇胺存在下火焰原子吸收光谱法测定钼

研究了在火焰原子吸收光谱法中三乙醇胺对钼的增感作用。在60g·L~(-1)三乙醇胺存在下,可使钼的吸光度增感25％,且能消除多种共存元素的干扰。建立了钼的质量分数大于0.4％的合金钢中钼的测定方法。     

以双硫酯为链转移剂的活性自由基聚合

合成并研究了两种双硫酯链转移剂的纯化方法 ,进行了多种单体以双硫酯为链转移剂的活性自由基聚合及嵌段共聚 .发现以PhC(S)SC(CH3) 2 Ph为链转移剂的效果比PhC(S)SCH(CH3)Ph好 ,聚合产物的多分散性系数较小 .引发剂与链转移剂的摩尔数比为 1∶3 5～ 1∶4 2时 ,得到多分散性系数小 ,实测分子量与理论分子量相近的聚合产物 .聚合物的分子量随时间和转化率的增加而增加 ,加入第二单体形成嵌段共聚物 ,具有活性聚合特征 .聚甲基丙烯酸酯大分子引发剂引发丙烯酸酯单体聚合时 ,聚合速度最快 .     

RADICAL GENERATION DURING THE ILLUMINATION OF AQUEOUS SUSPENSIONS OF TUNGSTEN OXIDE IN THE PRESENCE OF METHANOL, SODIUM FORMATE and SODIUM BICARBONATE. DETECTION BY SPIN TRAPPING

Abstract— Free radical intermediates appearing during illumination of aqueous suspensions of tungsten oxide were detected by electron spin resonance using the technique of spin trapping. Solutions irradiated contained methanol, formaldehyde, sodium formate and sodium hydrogen carbonate. Signals assigned to the spin adducts of the •H, •OH, •CO₂^- and •CH₂OH radicals were found.     

THE KINETICS OF THE DECOMPOSITION OF CYCLOHEXYL HYDROPEROXIDE IN THE PRESENCE OF VANADYL DIBENZOYLMETHANE

The kinetics of the decomposition of cyclohexyl hydroperoxide(CHHP) in benzene catalyzed by vanadyl dibenzoylmethane[V0(DBM).,] has been studied.It was found that the products of decomposition of CHHP were cyclohexanol and cyclohexanone,which are produced in about equimolar amount,and the product cyclohexanol obviously inhibited the decomposition of CHHP.The kinetics data can be satisfactorily described by the following equation (with [CHHP]0>>[VO(DBM)2]0)R0=kK[CHHP]0[VO(DBM)2]0/(1+k[CHHP]0)This is the kinetic evidence for the formation of a catalyst-hydro-peroxide intermediate.In the equation K is the stability constant of the catalyst-hydroperoxide intermediate complex;k is the rate constant for the decomposition of the complex.The rate constant K at 500℃ may be expressed as follows:k=1.9×108exp(-53.7×103/RT)S-1 with the activation energy Ea=53.7kJ mol-1