BACTERIORHODOPSIN AND THE SENSORY PIGMENT OF THE PHOTOSYSTEM 565 IN HALOBACTERIUM HALOBIUM

Abstract Blocking in vivo synthesis of retinal by addition of nicotine to the culture medium leads to the loss of photobehavior in Halobacterium halobium. Addition of rrans -retinol or frans-retinol₂ (3,4-dehy-droretinol) restores the responses to light decreases in the green-yellow spectral range. Action spectra of the reconstituted retinal- and retinal₂-photosystem show maximal sensitivity at 565 and 580 nm, respectively. Addition of retinol or retinol₂ also restores the formation of bacteriorhodopsin (BR) or bacteriorhodopsin₂ (BR₂= 3,4-dehydroretinal-bacterio-opsin complex). The absorption spectra of BR and BR₂, measured in isolated membranes, as well as in living bacteria, show maxima at 568 nm (BR) and at about 600 nm (BR₂), respectively. Comparison of the action spectrum of the retinal2-containing sensory photosystem with the absorption spectrum of BR₂ suggests that a retinal pigment different from BR is responsible for the photosensory behavior to green-yellow light.     

PHOTOSENSORY BEHAVIOR OF A BACTERIORHODOPSIN-DEFICIENT MUTANT, ET-15, OF Halobacterium halobium

– Halobacterium halobium , strain ET-15, which does not contain detectable amounts of bacteriorhodopsin (BR) shows behavioral responses to UV and yellow-green light. Attractant stimuli. i.e. light-increases in the yellow-green range or light-decreases in the UV, suppress the spontaneous reversals of the swimming direction for a certain time. Repellent stimuli, i.e. light-decreases in the yellow-green range or light-increases in the UV, elicit an additional reversal response after a few seconds. Action spectra of both sensory photosystems, PS 370 and PS 565, were measured with attractant as well as with repellent stimuli. As in BR-containing cells, maximal sensitivity was always found at 370 nm for the UV-system and at 565 nm for the long-wavelength system. Fluence-response curves at 370 and 565 nm obtained with strain ET-15 and with a BR-containing strain show that the sensitivity of both photosystems is not reduced in the absence of BR. It is concluded that BR is required neither for PS 565 nor for PS 370. Instead retinal-containing pigments different from BR have to be assumed to mediate photosensory behavior.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

TRANSFORMATION OF A BOP-HOP-SOP-I-SOP-II-Halobacterium halobium MUTANT TO BOP+: EFFECTS OF BACTERIORHODOPSIN PHOTOACTIVATION ON CELLULAR PROTON FLUXES AND SWIMMING BEHAVIOR

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H₂O₂ is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o-Di and tri-hydroxy groups of polyphenols undergo oxidation by a free-radical mechanism and a green intermediate anion-radical with absorption Δ_max= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a-hydroxy-methylperoxide formed from H₂O₂ and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D₂O by a factor 6.5. Quenchers of O₂(¹Δ_g) and 1,3-di-phenylisobenzofurane diminish light intensity in non-aqueous solutions. The data suggest ¹O₂ participation in the observed chemiluminescence. Thermo-chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi-enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.     

BEHAVIORAL RESPONSES IN Paramecium multimicronucleatum TO VISIBLE LIGHT

Specimens of colorless Paramecium multimicronucleatum were found to respond to visible light. They accumulated in the shaded region (photodispersal) of a half-shaded glass tube during 2 min exposure to visible light. The specimens showed avoiding reaction upon both spatial and temporal increase in light intensity (step-up photophobic response). Steady-state swimming velocity (orthokinesis) was higher, while steady-state frequency of spontaneous change in swimming direction (klinokinesis) was lower when the light intensity was kept higher. In a light with wavelength of 440 nm the velocity was highest, while the frequency was lowest. The specimens did not show phototaxis (light direction-oriented locomotion). Spectral sensitivity curves for both the photodispersal and the step-up photophobic response showed a major peak at 520 nm and a minor peak at 680 nm. The photodispersal seems to be caused mainly by the step-up photophobic response exhibited by the specimens at the dark-light border. The photokinetic responses enhance the degree of the photodispersal.     

Spectroscopic studies on bilirubin aggregate at liquid/liquid interface

Bilirubin (BR) aggregating at liquid/liquid interface was firstly detected by Fourier transform infrared (FTIR) imaging/spectroscopy combining with ultraviolet-visible (UV/Vis) absorption spectra. In the UV/Vis absorption spectra of BR aggregate, a new shoulder appeared at 474 nm, and BR absorption maximum underwent red shift from 450 nm to a longer wavelength at 497 nm, which indicates that BR aggregate was formed at the interface. Meanwhile, the BR molecule structure changed or conformation torsion, that is, the increase in orbit overlap or dihedral angle and the enhancement of exciton coupling. In the study of FTIR imaging/spectroscopy, the hydrogen bond-sensitive infrared bands of BR aggregate showed remarkable changes in band shift and intensity compared with those of BR powder, suggesting that the intramolecular hydrogen bonds broke out and internal structure changed. These new findings will be helpful for understanding of the BR molecular interaction, transportation, complex with serum albumin and metal ions, and the effect of BR aggregating on biomembrane and human tissues.

Cobalt Ion-Doped TiO(2) Photocatalyst Response to Visible Light

Photocatalytic activity under visible light irradiation was generated by doping a small amount of Co(2+) ions into TiO(2) particles. Nanometer-sized particles with the composition xCoO-(100-x) TiO(2) (xCo/TiO(2); 0300 nm) light irradiation but also induced the visible light (lambda>400 nm) response. The highest photocatalytic activities were obtained at x=0.03 for both irradiations. Copyright 2000 Academic Press.     

CHEMILUMINESCENCE IN THE PROCESS OF OXIDATIVE RING-OPENING OF PURPUROGALLINQUINONES

Abstract— The oxidation of purpurogalline (PPG) by alkaline solution of H₂O₂ pH 9–11 at 298°K is accompanied by chemiluminescence (CL) in the spectral range 400–600 nm with the maximum at 500 nm and quantum yield about 10^-6. The optimal concentrations of reactants with respect to maximal intensity are: 2 × 10^-4 M PPG, 10^-2 M NaOH, 1 M H₂O₂. Activation energy calculated from the maximum intensity of CL is 8.1×0.4 kcal/mole. Light emission occurs only when OH-groups of the phenolic ring of PPG undergo oxidation and the blue anion of o -PPG-quinone is formed. The rate that determines step in the reaction associated with luminescence is the nucleophilic attack of OOH^- ion on the blue anion of o -PPG-quinone. In this exergonic step (-ΔH = 63 to 230kcal/mole) the o - and/or p -quinone ring is opened and carbonyl derivatives of α-tropolone are produced. They display fluorescence in the region 400–600 nm. The fluorescence spectrum of the reaction mixture after oxidation of PPG is very close to that of CL. It is likely that carbonyl derivatives of α-tropolone are emitters of CL.     

PHOTOCHEMICAL BEHAVIOR OF BACTERIORHODOPSIN IMMOBILIZED IN NaCl PELLETS

Abstract— Some photochemical reactions of bacteriorhodopsin (BR) embedded in NaCl pellets (BR-NaCI) in the visible region are described here. BR in these preparations is a mixture of two classes of species: a drastically blue-shifted form and the unchanged purple pigment. Depending on the illumination history of the BR before being immobilized, both kinds of BR could be demonstrated in light-adapted (LA) and dark-adapted (DA) forms, but light adaptation was not possible once the pellets were made. Analogously to BR suspensions, the light-adapted blue-shifted BRexhibited an a/l-trans type photocycle, but the thermal steps were greatly slowed down (time constants 1 to 5 min). The parent species absorb at 506 nm. The DA blue-shifted BR exhibited absorption changes resolved into two photoreactions, one all-(rans- like (as in LA-BR) and another, 13-cw like, whose decay rate is also greatly slowed down (recovery time several hours). The parent species of the 13-cis like cycle absorb at 480 nm. That pigment fraction in the pellets whose absorption was not blue-shifted, also exhibited similar photoreactions to BR in suspension, but with an overall turnover rate only one order of magnitude slower. From a previous report (Lazarev and Terpugov, Biochim. Biophys. Acta 590 ,324–338,1980) and this one, it appears that the very slow photocycles in NaCl-BR of low moisture content originate from blue-shifted chromophores rather than from unchanged BR.     

光散射/荧光比率法测定全氟辛烷磺酸

在pH=1.81的Britton Robinson（BR）缓冲溶液中,全氟辛烷磺酸（PFOS）与罗丹明B（RhB）通过静电引力和疏水作用使RhB的光散射增强,荧光发生猝灭。 利用在普通荧光光度计上单次扫描获得的340 nm处的光散射强度（I340）与596 nm处的荧光强度（F596）之比,建立了测定PFOS的光散射/荧光比率法,检出限（3σ）为17 nmol/L。 方法用于环境水样中PFOS的测定,RSD≤4.2%。     

AgBr/TiO2复合催化剂:双注法制备及其可见光催化性能

以TiO₂为载体,采用双注法合成了AgBr/TiO₂复合光催化剂.利用X射线衍射仪(XRD)、紫外可见分光光度计(UV-Vis)和扫描电子显微镜(SEM)对AgBr/TiO₂的结构、光吸收、形貌进行了表征.研究了AgBr/TiO₂在可见光下对甲基橙和丁基罗丹明B的光催化活性.结果表明,与TiO₂相比,AgBr/TiO₂的光吸收范围拓展到400—600nm波段,并且随着AgBr负载量的增加,AgBr/TiO₂的可见光吸收强度增强;当AgBr与TiO₂的摩尔比为0.25时,AgBr/TiO₂具有最大催化活性;在相同条件下,AgBr/TiO₂对丁基罗丹明B的降解效果优于甲基橙.     

THE PHOTOREACTION OF THE DEIONIZED FORM OF THE PURPLE MEMBRANE INVESTIGATED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— Deionization of the purple membrane of Halobacterium halobium shifts the visible absorption maximum from 570 to 605 nm and inhibits proton transport. FTIR-difference-spectra of this blue membrane at 280 K reveal that the retinal chromophore adopts a 13 -cis and all -trans geometry in a light dependent ratio. In contrast to purple membrane the 13-cu isomer forms much faster in the dark. The all- trans component produces an L-intermediate which can be stabilized at 170 K. Spectral characteristics are similar to normal L. including comparable changes of internal aspartic acids of the opsin. However, stronger changes in the amide-I absorption are observed. IR bands of the chromo-protein states are assigned to retinal normal modes by the use of bacteriorhodopsin regenerated with'C-labeled retinals.     

纳米管钛酸的ESR特性及其可见光照的影响

本文报道了纳米管钛酸在真空-0.1MPa、温度100℃的条件下,经过不同时间处理后的ESR特性及其可见光照的影响.发现纳米管钛酸经一定处理后,不经光照即出现g=2.003ESR信号,该信号是由捕获一个电子的氧空位(V_o)产生的,此信号随着处理时间的延长而增强;在532nm的可见光照射下,随着光照时间的延长信号强度随之增加,达到一定强度值后,不再随光照时间的延长而增加;光源关闭后,信号强度又逐渐减小,但不能恢复到原来信号强度的水平.     

LASER PHOTOLYSIS OF THE PURPLE MEMBRANE OF Halobacterium halobium IN THE PHOTOSTATIONARY STATE: THE PHOTOBRANCHING PROCESS FROM THE O640 INTERMEDIATE

Abstract The photobranching process from the O₆₄₀ intermediate (O) in the photocycle of bacteriorhodopsin was studied. The O form accumulated with continuous wave visible light (390–800 nm) irradiation of the acidic (pH 3.9–6.0) purple membrane of Halobacterium halobium at 22°C. The photocycle of O via an L-like (or N-like) intermediate was driven by 630 nm pulsed light. The newly found intermediate has an absorption band in the 450–560 nm region. The "green-light"-absorbing pigment, tentatively called G₅₂₀, was converted to O with a time constant of (1.2 ± 0.2) ms. No M-like species was found in the cycle. The quantum yield of the cycle was estimated to be 0.30 ± 0.15.     

UPCONVERSION OF THE SINGLET OXYGEN 1269 nm EMISSION TO THE VISIBLE

Abstract— Experiments are described that enable the kinetic behavior of singlet oxygen, O₂(^IΔ_g), to be monitored in the time-resolved mode using a photomultiplier to detect deep orange light (γ_max 660 nm). This orange light is a consequence of the upconversion of the natural emission of O₂(^IΔ_g) at 1269 nm.     

Upconverting‐Nanoparticle‐Assisted Photochemistry Induced by Low‐Intensity Near‐Infrared Light: How Low Can We Go?

Upconverting nanoparticles (UCNPs) convert near‐infrared (NIR) light into UV or visible light that can trigger photoreactions of photosensitive compounds. In this paper, we demonstrate how to reduce the intensity of NIR light for UCNP‐assisted photochemistry. We synthesized two types of UCNPs with different emission bands and five photosensitive compounds with different absorption bands. A λ=974 nm laser was used to induce photoreactions in all of the investigated photosensitive compounds in the presence of the UCNPs. The excitation thresholds of the photoreactions induced by λ=974 nm light were measured. The lowest threshold was 0.5 W cm^?2, which is lower than the maximum permissible exposure of skin (0.726 W cm^?2). We demonstrate that low‐intensity NIR light can induce photoreactions after passing through a piece of tissue without damaging the tissue. Our results indicate that the threshold for UCNP‐ assisted photochemistry can be reduced by using highly photosensitive compounds that absorb upconverted visible light. Low excitation intensity in UCNP‐assisted photochemistry is important for biomedical applications because it minimizes the overheating problems of NIR light and causes less photodamage to biomaterials.     

Influence of Excitation Wavelength (UV or Visible Light) on the Photocatalytic Activity of Titania Containing Gold Nanoparticles for the Generation of Hydrogen or Oxygen from Water

Gold nanoparticles supported on P25 titania (Au/TiO(2)) exhibit photocatalytic activity for UV and visible light (532 nm laser or polychromatic light λ > 400 nm) water splitting. The efficiency and operating mechanism are different depending on whether excitation occurs on the titania semiconductor (gold acting as electron buffer and site for gas generation) or on the surface plasmon band of gold (photoinjection of electrons from gold onto the titania conduction band and less oxidizing electron hole potential of about -1.14 V). For the novel visible light photoactivity of Au/TiO(2), it has been determined that gold loading, particle size and calcination temperature play a role in the photocatalytic activity, the most active material (Φ(H2) = 7.5% and Φ(O2) = 5.0% at 560 nm) being the catalyst containing 0.2 wt % gold with 1.87 nm average particle size and calcined at 200 °C.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.