ABSTRACT
We have demonstrated the capability of a nanocomposite film made of a 2D array of Ag nanoparticles embedded into a poly(glycidyl methacrylate), PGMA, matrix to monitor the presence of organic vapors in the atmosphere. Specifically, changes in the extinction spectra of the submicron nanocomposite film are used to sense the vapors. The transformations of the spectra are fully reversible and reproducible upon multiple exposures. We associate this reversibility and reproducibility with the construction of the nanocomposite film where the cross-linked PGMA network is able to spatially restore its structure upon deswelling. The structure of the extinction spectrum of the film is governed by a collective surface plasmon mode excited in the Ag NPs array. It was found that spectral bands associated with normal and tangential components of the plasmon mode change their width and position when the nanocomposite is exposed to organic vapors. This is due to increasing the spacing between neighboring NPs and a decrease of the refractive index of the polymer caused by swelling of the PGMA matrix. Therefore, the level of spectral transformation is directly related to the level of polymer-solvent thermodynamic affinity where the higher affinity corresponds to the higher level of the swelling. Therefore, we expect that the nanocomposite films (when designed for a particular solvent) can be effectively used as a sensing element in a low-cost volatile organic compounds (VOC) sensor device operating in visual light.
ABSTRACT
Extinction spectra of 2D arrays of silver nanoparticles self-assembled on 5-20-nm-thick vacuum-deposited silver films exhibit extremely narrow plasmon mode in the blue spectral region, whereas the same particles self-assembled on thin films of Au, Cu, Cr and Ti damp the plasmon resonance. The observed narrowing was attributed to the plasmon coupling between the nanoparticles and between the nanoparticles and surface modes in the underlying silver film.
ABSTRACT
Surface-enhanced resonance Raman scattering (SERRS) spectra were measured for the beta-carotene and lycopene carotenoids present in low-density lipoproteins (LDLs), which were isolated from human plasma and adsorbed on roughened silver surfaces. The silver surface was modified by formation of a self-assembled monolayer (SAM) of carboxylate-terminated linear alkanethiols in order to simulate the LDL binding region of the cellular LDL receptor. Thiols of different chain length were used to produce SAMs of varying thicknesses. It was shown that carotenoids are not released from the LDL particle upon adsorption onto the bare and thiol modified silver surfaces. The SERRS studies indicated that beta-carotene and lycopene were present in the shell of the LDL particle. The dependence of SERRS on the distance from the silver surface was different for beta-carotene and lycopene in LDL. This observation suggests that the two carotenoids are located in different places of the LDL particle.
Subject(s)
Carotenoids/metabolism , Lipoproteins, LDL/blood , Antioxidants/chemistry , Antioxidants/metabolism , Carotenoids/chemistry , Humans , Lipoproteins, LDL/chemistry , Lycopene , Spectrum Analysis, Raman , Surface Plasmon Resonance , beta Carotene/chemistry , beta Carotene/metabolismABSTRACT
Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.
Subject(s)
Ectothiorhodospira/chemistry , Photosynthetic Reaction Center Complex Proteins/chemistry , Rhodobacter sphaeroides/chemistry , Spectrum Analysis, Raman/methods , Light-Harvesting Protein ComplexesABSTRACT
The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms. The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed. From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT. Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT. Similar distortions are present in the reduced form of the proteins. Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.
Subject(s)
Amino Acid Substitution , Cytochromes c1/chemistry , Cytochromes c1/genetics , Circular Dichroism , Ligands , Oxidation-Reduction , Point Mutation , Porphyrins/chemistry , Protein Structure, Tertiary , Spectrum Analysis, Raman , Yeasts/chemistryABSTRACT
The excited-state intramolecular H-atom transfer reactions of hypocrellins B and A are compared by using time-resolved absorption and fluorescence upconversion techniques. The hypocrellin B photophysics are well described by a simple model involving one ground-state species and excited-state forward and reverse H-atom transfer with a nonfluorescent excited state. We suggest that excited-state conformational changes are coupled to the H-atom transfer in hypocrellin B just as gauche/anti changes are coupled to the H-atom transfer in hypocrellin A.
Subject(s)
Perylene/analogs & derivatives , Photosensitizing Agents/chemistry , Quinones/chemistry , Kinetics , Molecular Structure , Perylene/chemistry , Phenol , Photochemistry , Solvents , SpectrophotometryABSTRACT
omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole). The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM. The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles. Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM). Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM). Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility. The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32. 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3. NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide. Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.
Subject(s)
Bacterial Proteins , Cytochrome P-450 Enzyme System/chemistry , Fatty Acids/chemistry , Imidazoles/chemistry , Mixed Function Oxygenases/chemistry , Arachidonic Acid/metabolism , Binding Sites , Catalysis , Circular Dichroism , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Fatty Acids/metabolism , Heme/chemistry , Heme/metabolism , Imidazoles/metabolism , Kinetics , Lauric Acids/metabolism , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , NADP/metabolism , NADPH-Ferrihemoprotein Reductase , Protein Structure, Tertiary , Spectrophotometry, Ultraviolet , Substrate SpecificityABSTRACT
Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir--Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin--fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating layers of bovine serum albumin--biotin conjugate (BSA--biotin) and avidin. The dependence of fluorescence intensity on the number of lipid or protein spacer layers deposited on the surface of the CMF was investigated. The results demonstrate the requirement for adsorbate location within the region between Ag particles for maximal enhancement. The density of avidin molecules on the surface of the BSA--biotin/avidin multilayers adsorbed on the CMF was also determined. A procedure for forming a rigid, uniform silica layer around the Ag particles on the CMF is described. The layer protects the particles from undesirable chemical reactions such as etching by halide ions, for example, and provides the requisite stability for bioanalytical applications. Colloidal films composed of Ag particles covered by approximately 10-nm-thick silica layers were tested for fluorescence enhancement using goat immunoglobulin and a conjugate of rabbit anti-goat immunoglobulin with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)hexanoate. An enhancement factor of approximately 20 was obtained.
Subject(s)
Colloids/chemistry , Metals/chemistry , Animals , Fluorescence , Goats , Immunoglobulins/analysis , Microscopy, Electron, Scanning , Rabbits , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.
Subject(s)
Photosynthetic Reaction Center Complex Proteins/chemistry , Photosystem II Protein Complex , Pressure , Thermodynamics , X-Ray DiffractionABSTRACT
Surface-enhanced Raman spectra of native and photobleached bovine rod outer segment disks as well as inside-out (inverted) photoreceptor disks adsorbed on silver hydrosol have been analyzed. Surface-enhanced spectra of inverted disks and disk-monoclonal antibody complexes reveal the short-range mechanism of enhancement. The distance between retinal Schiff base and the cytoplasmic side of native disk has been shown to be 5-10 A.
Subject(s)
Photoreceptor Cells/metabolism , Retinaldehyde/metabolism , Retinoids/metabolism , Animals , Cattle , Halobacterium , Microscopy, Electron , Rod Cell Outer Segment/metabolism , Spectrum Analysis, Raman , Sulfhydryl CompoundsABSTRACT
Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.