Biospectroscopy最新文献

We describe spatially localized two-photon excitation in scattering media. Using femtosecond pulses at 770 nm from a Ti : Sapphire laser, we were able to excite fluorophores in capillary tubes under up to 1.5 mm of 0.5% intralipid. Displacement of the laser beam relative to the embedded samples indicates that highly localized excitation was possible with two-photon excitation, whereas one-photon excitation resulted in loss of spatial resolution due to excitation by the diffusely scattered photons. These results indicate that two-photon excitation in the scattering solution is due only to the ballistic photons, a result confirmed by frequency-domain time-resolved measurements. Selective excitation of adjacent embedded samples was found possible for two but not one-photon excitation. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 303–310, 1998

The terminal caa₃ oxidase of Thermus thermophilus has been studied by resonance Raman spectroscopy. Using different excitation wavelengths in the Soret band region, it was possible to disentangle the resonance Raman spectra of the fully oxidized and fully reduced state in terms of the component spectra of the individual hemes a, a₃, and c. For the heme a and a₃ groups, the spectra reveal only minor differences compared to those of beef heart cytochrome c oxidase attributable to subtle modifications of the heme environment. These differences are not more pronounced than those between the oxidases from beef heart and Paracoccus denitrificans confirming the view that this oxidase of Th. thermophilus is a typical member of the aa₃ oxidase superfamily. The heme c component spectra display far-reaching similarities with those of c-type cytochromes which serve as mobile electron carriers in the respiratory chain. These results imply that caa₃ oxidase represents an integrated version of the noncovalent redox complex between cytochrome c and cytochrome c oxidase in higher organisms. On the other hand, the structural changes of cytochrome c in the noncovalent complex have no counterpart in the heme c component of the caa₃ oxidase indicating a specific cytochrome c binding site for the mitochondrial enzyme. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 365–377, 1998

Electronic absorption and resonance Raman spectra of various peroxidases and selected site-directed mutants are reported. These results and the X-ray crystal structure data are critically analyzed and underline the differences that exist between the crystal and solution states. The effect of the vinyl conjugation on the electronic absorption maxima and the influence of the ligand nature on the wavelength of the charge-transfer (CT1) band are shown to be useful probes of subtle interactions in the heme pocket. The spectroscopic differences observed between the three classes of peroxidases are discussed in terms of their structural diversity. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S3–S17, 1998

We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm⁻¹. The strong NH combination band found at 4867 cm⁻¹ in the spectrum of native RNase A shifts to 4878 cm⁻¹ upon thermal unfolding. Hydrogen–deuterium exchange experiments that validate the NH character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D₂O. The transition profiles and temperatures derived from the temperature dependence of the NH combination mode were found to be practically identical with those derived from the temperature dependence of the CO amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H₂O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in β-sheet structure, followed by the loss of α-helical structures, and then ending with the unfolding of the remaining β-sheets. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S19–S29, 1998

¹³C- and ⁵⁷Fe-NMR spectra of several carbon monoxide hemoprotein models with varying polar and steric effects of the distal organic superstructure, constraints of the proximal side, and solvent polarity are reported. The ¹³C shieldings of heme models cover a 4.0 ppm range that is extended to 7.0 ppm when several hemoglobin CO and myoglobin CO species at different pHs are included. Both heme models and heme proteins obey a similar excellent linear δ(¹³C) versus ν(CO) relationship that is primarily due to modulation of π backbonding from Fe d_π to the CO π* orbital by the distal pocket polar interactions. There is no direct correlation between δ(¹³C) and FeCO geometry. The poor monotonic relation between δ(¹³C) and ν(FeC) indicates that the iron-carbon π bonding is not a primary factor influencing δ(¹³C) and δ(⁵⁷Fe). The δ(⁵⁷Fe) was found to be extremely sensitive to deformation of the porphyrin geometry, and increased shielding by more than 600 ppm with increased ruffling was observed for various heme models of known X-ray structures. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S57–S69, 1998

A molecular force field dedicated to molecular dynamics simulation of biomembranes was developed. It was parameterized on model compounds related to phospholipids and was able to reproduce at the same time structures, energies, and vibrational spectra. Cross terms in the potential energy function were introduced by solving the redundancy problem among internal coordinates. This force field was used in the 400-ps molecular dynamics simulation of a hydrated bilayer in the gel and liquid crystal phases. The conformational properties of the polar head groups were in particular agreement with the experimental observations using Raman scattering. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S41–S46, 1998

An optimized FT Raman microscope (inverted microscope with high throughput of radiation) was developed that allows minimal sample preparation and Raman spectroscopy without fluorescence. A quantitative determination of the mineralization of bone tissue and hydroxyapatite (HA) coatings of hip and knee prostheses was performed. The lateral resolution reached down to 10 μm. The distribution of the HA content in the coatings investigated was found to be similar all the time. This result was independent of the composition of the coatings and the history of the whole prosthesis. In the immediate vicinity of the prosthesis a large HA content could be observed that decreased to a minimum towards the periphery of the coating and increased at the site of the ongrown bone. For the interface between bone and HA coating a transitional zone was observed at a lateral distance of 30–40 μm to the implant. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 403–412, 1998

Gold and silver vacuum-deposited island films were characterized by studying deposition variables such as film thickness, evaporation rate, and substrate temperature. For both metals, these parameters were correlated with the surface-enhanced Raman spectroscopy (SERS) effect and an increase in film thickness and low evaporation rates were shown to upshift the wavelength at maximum optical density (λ_max) and increase the optical density of the substrates. In contrast, pre- and postdeposition annealing of gold films led to the formation of substrates that exhibited a downshift of λ_max. Our spectral data also indicated that silver films are substrates that are more suited for SERS applications where high frequency visible excitations are used. Measurements on gold films classified them into two groups: thin Au films (10–50 Å) well adapted for red excitations and thicker ones that are operative in the near infrared. SERS results, which were obtained from a single HL60 cell treated with micromolar drug quantities, placed on thin gold island films indicated that these island films could be future promising substrates for SERS imaging at the cellular level. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: S71–S78, 1998

Hemoglobin I (HbI) from the claim Lucina pectinata is a unique heme protein that binds and transfers hydrogen sulfide (H₂S) to a symbiotic bacteria. The metcyano, metaquo, carbon monoxy, oxy, and deoxy complexes of HbI were studied by resonance Raman (RR) spectroscopy, and the metcyano and carbon monoxy complexes were also studied by ¹H-NMR. The results indicate a unique orientation of the heme 2-vinyl group relative to other heme proteins. The RR spectra of the HbICO, metHbICN, metHbIH₂O, HbIO₂ and deoxyHbI heme derivatives show a band at 1621 cm⁻¹ and a shoulder at 1626 cm⁻¹, indicative of an out-of-plane position for one of the vinyls relative to the other one. Spin-lattice relaxation properties of protons in the metHbICN complex also suggest a unique orientation for the heme 2-vinyl group of HbI. The longitudinal relaxation time (T₁) for the 2-H_α, 2-H_βc, and H_βt protons are 120 ms, 115 ms, and 135 ms, respectively. The data from both techniques suggest an out-of-plane and trans-oriented 2-vinyl group, and an in-plane and cis-oriented 4-vinyl group for the low-spin complexes of HbI. These results imply that the electron withdrawing character of the out-of-plane vinyl group contributes to the stability of the heme Fe⁺³ oxidation state, facilitates the binding of the H₂S ligand, and promotes the stability of this ferric H₂S complex. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 311–326, 1998

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) have been applied to study the aggregation and excimer emission of 9-aminoacridine (9AA) and 9-aminoacridine hydrochloride (9AA-HCl) in aqueous solution and on silver colloids. The effect of the drug concentration, pH, and chloride concentration on these processes has been investigated. The excimer emission of 9AA is connected to the dimerization of this drug in solution: the formation of 9AA dimers is greatly favored when the drug is under the amino form at neutral and acidic pH, while at alkaline pH the imino 9AA form tends to form large-sized aggregates which cannot be excited to render excimer emission. 9AA is adsorbed on the silver surface under two different forms: strongly and weakly attached 9AA, each one corresponding to the different drug tautomers: imino and amino. The interaction of 9AA with silver induces a charge transfer from the adsorbate to the metal leading to a remarkable fluorescence quenching, a basicity decrease of the adsorbed drug and a considerable weakening of the dimer-excimer emission. Furthermore, an attribution of the main Raman features appearing in the SERS spectra has been proposed, providing marker bands for the imino and amino 9AA tautomers, and a mechanism for the molecular dimerization is also suggested. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 327–339, 1998