RESUMO
In oxygenic plants, photons are captured with high quantum efficiency by two specialized reaction centers (RC) called Photosystem I (PS I) and Photosystem II (PS II). The captured photon triggers rapid charge separation and the photon energy is converted into an electrostatic potential across the nanometer-scale (~6 nm) reaction centers. The exogenous photovoltages from a single PS I RC have been previously measured using the technique of Kelvin force probe microscopy (KFM). However, biomolecular photovoltaic applications require two-terminal devices. This paper presents for the first time, a micro-device for detection and characterization of isolated PS I RCs. The device is based on an AlGaN/GaN high electron mobility transistor (HEMT) structure. AlGaN/GaN HEMTs show high current throughputs and greater sensitivity to surface charges compared to other field-effect devices. PS I complexes immobilized on the floating gate of AlGaN/GaN HEMTs resulted in significant changes in the device characteristics under illumination. An analytical model has been developed to estimate the RCs of a major orientation on the functionalized gate surface of the HEMTs.
Assuntos
Técnicas Biossensoriais , Complexo de Proteína do Fotossistema I/química , Transistores Eletrônicos , Alumínio/química , Elétrons , Gálio/química , Ouro/química , Mercaptoetanol/química , Microscopia de Força Atômica , Nitrogênio/química , Fótons , Espectrofotometria UltravioletaRESUMO
PURPOSE: To study the feasibility of anterior vitreal oxygenation for the treatment of acute retinal ischemia. METHODS: Twenty rabbits were randomized into an oxygenation group, a sham treatment group, and a no treatment group. Baseline electroretinography (ERG) and preretinal oxygen (Po(2)) measurements were obtained 3 to 5 days before surgery. Intraocular pressure was raised to 100 mm Hg for 90 minutes and then normalized. The oxygenation group underwent vitreal oxygenation for 30 minutes using intravitreal electrodes. The sham treatment group received inactive electrodes for 30 minutes while there was no intervention for the no treatment group. Preretinal Po(2) in the posterior vitreous was measured 30 minutes after intervention or 30 minutes after reperfusion (no treatment group) and on postoperative days (d) 3, 6, 9, and 12. On d14, rabbits underwent ERG and were euthanatized. RESULTS: Mean final (d12) Po(2) was 10.64 ± 0.77 mm Hg for the oxygenation group, 2.14 ± 0.61 mm Hg for the sham group, and 1.98 ± 0.63 mm Hg for the no treatment group. On ERG, scotopic b-wave amplitude was significantly preserved in the oxygenation group compared with the other two groups. Superoxide dismutase assay showed higher activity in the operated eyes than in the nonoperated control eyes in the sham treatment group and no treatment group only. Histopathology showed preservation of retinal architecture and choroidal vasculature in the oxygenation group, whereas the sham-treated and nontreated groups showed retinal thinning and choroidal atrophy. CONCLUSIONS: In severe total ocular ischemia, anterior vitreal oxygenation supplies enough oxygen to penetrate the retinal thickness, resulting in rescue of the RPE/choriocapillaris that continues to perfuse, hence sparing the retinal tissue from damage.
Assuntos
Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Doenças Retinianas/metabolismo , Corpo Vítreo/metabolismo , Animais , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Pressão Intraocular , Microscopia Eletrônica de Varredura , Coelhos , Retina/enzimologia , Superóxido Dismutase/metabolismoRESUMO
Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.
Assuntos
Potenciais da Membrana/efeitos da radiação , Complexo de Proteína do Fotossistema I/efeitos da radiação , Arabidopsis , Concentração de Íons de Hidrogênio , Luz , Modelos Biológicos , Modelos Químicos , Proteolipídeos , Prótons , Valinomicina/farmacologiaRESUMO
This research identified toxin detection limits using the variable fluorescence of naturally occurring microalgae in source drinking water for five chemical toxins with different molecular structures and modes of toxicity. The five chemicals investigated were atrazine, Diuron, paraquat, methyl parathion, and potassium cyanide. Absolute threshold sensitivities of the algae for detection of the toxins in unmodified source drinking water were measured. Differential kinetics between the rate of action of the toxins and natural changes in algal physiology, such as diurnal photoinhibition, are significant enough that effects of the toxin can be detected and distinguished from the natural variance. This is true even for physiologically impaired algae where diminished photosynthetic capacity may arise from uncontrollable external factors such as nutrient starvation. Photoinhibition induced by high levels of solar radiation is a predictable and reversible phenomenon that can be dealt with using a period of dark adaption of 30 minutes or more.
Assuntos
Monitoramento Ambiental , Eucariotos/isolamento & purificação , Água Doce , Poluentes Químicos da Água/química , Herbicidas/química , Abastecimento de ÁguaRESUMO
This communication discloses new ideas and preliminary results on the development of a metabolic prosthesis for local oxygenation of ischemic tissue under physiologically neutral conditions. We report for the first time selective electrolysis of physiological saline by repetitively pulsed, charge-limited electrolysis for the production of oxygen and suppression of free chlorine. Using 800-microA amplitude current pulses and < 200 micros pulse duration, we demonstrate prompt oxygen production and delayed chlorine production at the surface of a fused 0.85-mm diameter spherical platinum electrode. The data, interpreted in terms of the ionic structure of the electric double layer, suggest a strategy for in situ production of metabolic oxygen via a new class of "smart" prosthetic implants for ischemic disease such as diabetic retinopathy. We also present data indicating that collateral pH drift, if any, can be held constant using a feedback-controlled three-electrode electrolysis system that chooses an anode and cathode pair based on pH data provided by a local sensor.
Assuntos
Técnicas Eletroquímicas , Eletrodos Implantados , Isquemia/terapia , Oxigênio/metabolismo , Próteses e Implantes , Simulação por Computador , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Órbita , Desenho de Prótese , Cloreto de Sódio/química , Cloreto de Sódio/metabolismo , Água/química , Água/metabolismoRESUMO
Small-angle X-ray scattering (SAXS) was used to investigate the structure of isolated photosystem I (PSI) complexes stabilized in detergent solution. Two different types of PSI preparation were investigated. In the first preparation, thylakoid membranes were solubilized with Triton X100 and purified by density gradient centrifugation. SAXS data indicated large scattering objects or microphases that can be described as sheets with approximately 68 A thickness and a virtually infinite lateral extension. The observed thickness agreed well with the dimension of a PSI molecule across the thylakoid membrane. In the second preparation, PSI was isolated as before but was further purified by anion exchange chromatography resulting in functional complexes consisting of single PSI units with attached surfactant as evidenced by the particle volume and gyration radius extracted from the SAXS data. Several approaches were used to model the solution conformation of the complex. Three different ellipsoidal modeling approaches, a uniform density ellipsoid of revolution, a triaxial solid ellipsoid, and a core-shell model, found extended structures with dimensions that were not consistent with the PSI crystal structure (Ben-Shem, A.; et al. Nature 2003, 426, 630-635). Additionally, the SAXS data could not be modeled using the crystal structure embedded in a disk of detergent. The final approach considered the possibility that protein was partially unfolded by the detergent. The data were modeled using a "beads-on-a-string" approach that describes detergent micelles associated with the unfolded polypeptide chains. This model reproduced the position and relative amplitude of a peak present in the SAXS data at 0.16 A(-1) but was not consistent with the data at larger length scales. We conclude that the polypeptide subunits at the periphery of the PSI complex were partially unfolded and associated with detergent micelles while the catalytically active core of the PSI complex remained structurally intact. This interpretation of the solution structure of isolated PSI complexes has broader implications for the investigation of the interactions of detergents and protein, especially for crystallization studies.
Assuntos
Detergentes/química , Proteínas de Membrana/química , Complexo de Proteína do Fotossistema I/química , Cristalização , Eletroforese em Gel de Poliacrilamida , Espalhamento de Radiação , Raios XRESUMO
We report the direct measurement of photoinduced surface potential differences of wild-type (WT) and mutant D96N bacteriorhodopsin (BR) membranes at pH 7 and 10.5. Atomic force microscopy (AFM) and scanning surface potential microscopy (SSPM) were used to measure the BR membrane with the extracellular side facing up. We present AFM and SSPM images of WT and mutant D96N in which the light-dark transition occurred in the mid-scan of a single BR membrane. Photosteady-state populations of the M state were generated to facilitate measurement in each sample. The photoinduced surface potential of D96N is 63 mV (peak to valley) at pH 10.5 and is 48 mV at pH 7. The photoinduced surface potential of WT is 37 mV at pH 10.5 and approximately 0 at pH 7. Signal magnitudes are proportional to the amount of M produced at each pH. The results indicated that the surface potentials were generated by photoformation of surface charges on the extracellular side of the membrane. Higher surface potential correlated with a longer lifetime of the charges. A mechanistic basis for these signals is proposed, and it is concluded that they represent a steady-state measurement of the B2 photovoltage.
Assuntos
Bacteriorodopsinas/química , Mutação , Bacteriorodopsinas/genética , Microscopia de Força Atômica , Modelos Moleculares , FotoquímicaRESUMO
Photosynthetic reaction centers are integral membrane complexes. They have potential application as molecular photovoltaic structures and have been used in diverse technological applications. A three-dimensional electrostatic model of the photosystem I reaction center (PSI) embedded in a lipid membrane is presented. The potential is obtained by solving the Poisson-Boltzmann equation with the finite element method (FEM). Simulations showing the potential distribution in a vesicle containing PSI reaction centers under different conditions are presented. The results of the simulations are compared with previous findings and a possible application of PSI to provide light activation of voltage-gated ion channels is discussed.
Assuntos
Fotossíntese/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/química , Cinética , Lipossomos/química , Modelos Moleculares , Complexo de Proteína do Fotossistema I/química , Distribuição de Poisson , Eletricidade EstáticaRESUMO
Photosynthetic reaction centers are integral plant membrane protein complexes and molecular photovoltaic structures. We report here that addition of Photosystem I (PSI)-proteoliposomes to retinoblastoma cells imparts photosensitivity to these mammalian cells, as demonstrated by light-induced movement of calcium ions. Control experiments with liposomes lacking PSI demonstrated no photosensitivity. The data demonstrate that PSI, a nanoscale molecular photovoltaic structure extracted from plants, can impart a photoresponse to mammalian cells in vitro.
Assuntos
Sinalização do Cálcio/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/efeitos da radiação , Folhas de Planta/metabolismo , Retina/metabolismo , Retina/efeitos da radiação , Spinacia oleracea/metabolismo , Sinalização do Cálcio/efeitos da radiação , Linhagem Celular , Relação Dose-Resposta à Radiação , Humanos , Luz , Fotobiologia/métodos , Doses de RadiaçãoRESUMO
A method of imaging nanometer metallocatalysts formed by photosynthetic precipitation of the water-soluble transition-metal compounds [PtCl(6)](2-) and [RuCl(6)](2-) is reported. Hexachloroplatinate and hexachlororuthenate can accept up to four electrons from Photosystem I (PSI) reaction centers in photosynthetic thylakoid membranes, thereby converting [PtCl(6)](2-) and [RuCl(6)](2-) anions to either metallic platinum (Pt) and ruthenium (Ru) and/or partially oxidized nanometer catalysts at the reducing sides of PSI molecules. Use of this method can potentially create nanometer-sized Pt and/or bimetallic catalysts (such as Pt-Ru) on biomembranes and molecules at pH 7 and room temperature with preservation of the biological function of the molecules.
Assuntos
Fotossíntese/fisiologia , Elementos de Transição/química , Água/química , Catálise , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Platina/química , Rutênio/química , Tilacoides/química , Tilacoides/metabolismo , Tilacoides/ultraestruturaRESUMO
We have used self-assembled monolayers (SAMs) prepared from omega-terminated alkanethiols on gold to generate model surfaces and examine the effect of surface composition on the adsorption of Photosystem I (PSI), stabilized in aqueous solution by Triton X-100. Triton-stabilized PSI adsorbs to high-energy surfaces prepared from HO- and HO2C-terminated alkanethiols but does not adsorb to low-energy surfaces. The inhibition of PSI adsorption at low-energy surfaces is consistent with the presence of a layer of Triton X-100 that adsorbs atop the hydrophobic SAM and presents a protein-resistant poly(ethylene glycol) (PEG) surface. While the presence of the PEG surface prevents the adsorption of PSI, the displacement of the inhibiting layer of Triton X-100 by dodecanol, a more active surfactant, greatly enhances the adsorption of PSI. This inhibiting effect by Triton X-100 can be extended to other protein systems such as bovine serum albumin.
RESUMO
We have discovered a new competitive pathway for O2 sensitivity in algal H2 production that is distinct from the O2 sensitivity of hydrogenase per se. This O2 sensitivity is apparently linked to the photosynthetic H2 production pathway that is coupled to proton translocation across the thylakoid membrane. Addition of the proton uncoupler carbonyl cyanide-p-trifluoromethoxy- phenylhydrazone eliminates this mode of O2 inhibition on H2 photoevolution. This newly discovered inhibition is most likely owing to background O2 that apparently serves as a terminal electron acceptor in competition with the H2 production pathway for photosynthetically generated electrons from water splitting. This O2-sensitive H2 production electron transport pathway was inhibited by 3[3,4-dichlorophenyl]1,1-dimethylurea. Our experiments demonstrated that this new pathway is more sensitive to O2 than the traditionally known O2 sensitivity of hydrogenase. This discovery provides new insight into the mechanism of O2 inactivation of hydrogenase and may contribute to the development of a more-efficient and robust system for photosynthetic H2 production.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Eucariotos/metabolismo , Hidrogênio/metabolismo , Oxigênio/análise , Fotossíntese , Animais , Hidrogênio/análise , Sensibilidade e EspecificidadeRESUMO
Working with primary-source freshwater drinking samples from the Clinch and Tennessee Rivers, we have developed a tissue-based biosensor detection system that uses naturally occurring aquatic photosynthetic tissue as the sensing material for detection of chemical antagonists in the water. Sensor readout is based on well-known principles of fluorescence induction by living photosynthetic tissue. The Clinch River is the main source of drinking water for Oak Ridge, Tennessee, while the Tennessee River is a major source for the city of Knoxville. We have successfully detected algae in every sample that we examined and readily monitored changes in the characteristic fluorescence induction curves when the samples were exposed to potassium cyanide (KCN), methyl parathion (MPt), N'(3,4-dichlorophenyl)-N,N-dimethylurea (DCMU), and paraquat. The percentage decreases in photochemical yields observed in Tennessee River samples after a 24-min exposure to KCN, MPt, and DCMU were, respectively, 21.89+/-0.76, 3.28+/-0.18, and 14.77+/-1.81. For a site at the Clinch River, the percentage decreases were 22.78+/-1.63, 8.32+/-0.21, and 17.71+/-1.32 (Table 1). The unique aspect of this approach to real-time water quality monitoring is that unlike conventional sensing devices, this sensor material is external to the detecting instrument and is continuously refreshed. These biosensors may be used as continuous rapid-warning sentinels for detection of chemical warfare agents in sunlight-exposed drinking water supplies.