Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.     

Raman spectra of somatic nerves under different functional conditions

The light scattering spectra (LS) of different kind of somatic nerves during excitation and its block has been investigated. In the LS spectra of frog nerves the spectra of C40-carotenoids was dominated by the following bands at 1008 cm-1, 1160 cm-1 and 1526 cm-1. The peaks of LS were decreased during the excitation and under the action of ouabain and increased under the action of ether. The changes of LS spectra can be explained by shift of maximum of light intensity of C40-carotenoids uptake and positions of side CH3-radical in polyene chain.     

A study of the thermal stability of the Z form of (m5dC-dG)3 by resonance Raman spectroscopy.

The thermal stability of the hexanucleoside pentaphosphate m5dCpdGpm5dCpdGpm5 dCpdG has been studied by resonance Raman spectroscopy with 257 nm excitation wavelength. At low temperature and in 3M NaClO4, the Raman spectrum resembles that of poly(dG-dC).poly(dG-dC) in the Z conformation. As the temperature is increased, the position and the intensity of several bands (1312 cm-1, 1482 cm-1, 1584 cm-1 and 1632 cm-1) are modified. The variation of intensity versus temperature is biphasic. Analysis of the results suggests that the increase of temperature induces first a transition from the Z form to an intermediate stable form which then melts. These results and those previously obtained by circular dichroism and 31P nuclear magnetic resonance suggest that the intermediate form belongs to the left family but with changes in the stacking of the bases and the geometry of the phosphate groups as compared to the canonical Z form.     

Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.     

Low levels of irradiation modify lipid domains in model membranes: a laser Raman study

We have used Raman spectroscopy to study the effects of ionizing radiation on thermal transitions of dipalmitoyl lecithin + polyunsaturated fatty acid liposomes. Raman spectra in the CH (2800-3000 cm-1), C = C (1600-1680 cm-1), and C-C (1000-1150 cm-1) stretching regions are sensitive to ionizing radiation. The CH stretching of acyl chains yields three strong bands around 2850, 2880, and 2930 cm-1. The ratios of the relative intensities of 2880 and 2850 cm-1 bands, i.e., I2880/2850, when plotted against temperature show multiple infection points which correspond to multiple spectroscopic transitions. These are ascribed to a separate phase with distinctive proportions of lecithin and polyunsaturated fatty acids. We find these transitions sensitive to low levels of ionizing radiation. Doses as low as 5-15 rad after 48 h of 60Co gamma irradiation and 60 kVp X irradiation drastically broaden and shift the polyunsaturated rich phase which occurs at lower temperatures (-7 to +5 degrees C) than that of pure dipalmitoyl lecithin (39 degrees C). In addition a new transition around 46 degrees C also emerges upon irradiation (48 h postirradiation). These irradiation effects can be accelerated by the presence of catalytic amounts of Fe2+/EDTA +H2O2. The membrane transition modification is more sensitive to 60 kVp X rays in comparison to 60Co gamma rays owing to the high LET component of the former. The intensity of 1660 cm-1 band, assigned to C = C stretching in the cis-configuration, loses intensity upon irradiation. Concomitantly, a new band around 1675 cm-1, assigned to trans-configuration, emerges. Similarly the increase in the "order parameter" as calculated from the relative intensities of C--C stretching bands indicates rigidification of membrane. Various factors such as reduction in unsaturation, increase in trans-configuration, and the formation of multiple peroxidation products are invoked as lipid phase modifiers.     

Resonance Raman studies of the flavin and iron-sulfur centers of milk xanthine oxidase

Resonance Raman spectroscopy has been used to study milk xanthine oxidase, an enzyme containing molybdenum, binuclear iron-sulfur clusters, and FAD as cofactors. The contribution of FAD dominates the resonance Raman spectrum at frequencies above 500 cm-1. As expected, no bands assignable to FAD are observed in deflavo xanthine oxidase. The resonance Raman spectrum below 500 cm-1 reveals the contribution of the Fe2S2(Cys)4 groups with frequencies similar to those of adrenodoxin and putidaredoxin. Resonance enhancement profiles of the Fe2S2(Cys)4 clusters indicate intensity variations among the Fe2S2(Cys)4 peaks that are attributed to different excitation wavelength maxima of their bridging and terminal iron-sulfur vibrations. No evidence for Mo-ligand vibrations could be obtained by using excitation wavelengths between 363.8 and 514.5 nm.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm^?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm^?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H₂O and in ²H₂O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm^?1 attributable to membrane-associated carotenoids.     

Resonance Raman spectroscopy of horseradish peroxidase derivatives and intermediates with excitation in the near ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.     

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.     

A resonance Raman study on the reaction intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra of two reaction intermediates of D-amino acid oxidase with substrate analogs were obtained. The reaction intermediates studied were (1) the one in the aerobic oxidative reaction of the enzyme with beta-cyano-D-alanine and (2) the other in the reverse reductive reaction of the enzyme with chloropyruvate and ammonium. Both intermediates are characterized with the charge transfer absorption bands in the long wavelength region extending beyond 600 nm. The RR spectra of the two intermediates excited at 488.0 or 514.5 nm are those of oxidized flavin, which is consistent with our previous assumption that oxidized flavin is involved in these reaction intermediates. Relatively simple RR spectra were obtained for these intermediates with excitation at 632.8 nm which is within the region of the charge transfer bands. The resonance enhancement for the Raman lines around 1585 and 1350 cm-1 for either of the intermediates with excitation in the region of the charge transfer bands suggests that the charge transfer interaction involves the N(5)-C(4a) region extending to the C(10a)-N(1)-C(2) region of the isoalloxazine nucleus. The Raman line at 1657 cm-1 for the intermediate with chloropyruvate and ammonium was assigned to C = N of an imino acid from the isotopic frequency shift upon 15N-substitution. The assignment substantiates our previous conclusion that the intermediate involves an imino acid, alpha-imino-beta-chloropropionate.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

一种酵母细胞生长现象的实时单细胞拉曼光谱观察

用拉曼镊子观察单个即发活性干酵母(Saccharomy cescerevisiae)细胞在2.0%葡萄糖溶液中的活化过程,收集其拉曼光谱。结果发现,在某一批次的产品中,酵母细胞的1364cm-1峰强度随着细胞的活化而显著增加,531cm-1、652cm-1、1053cm-1等源自葡萄糖或葡萄糖基的信号峰也会随细胞的生长而增强,随后增强的还有1432cm-1、1448cm-1、1561cm-1等源自脂类物质的峰,而源自蛋白质及脂类的1000cm-1、1445cm-1、1655cm-1等峰的信号强度基本不变,酵母细胞代谢活跃的标志峰1603cm-1也基本不变。该批次产品中,10次实验有7次观察到上述现象,而在别的批次产品中并没有观察到该现象。用单细胞拉曼光谱实时记录了这一特殊的生长现象。     

Primary donor structure and interactions in bacterial reaction centers from near-infrared Fourier transform resonance Raman spectroscopy

Preresonance Raman and resonance Raman spectra of the primary donor (P) from reaction centers of the Rhodobacter (Rb.) sphaeroides R26 carotenoidless strain in the P and P+ states, respectively, were obtained at room temperature with 1064-nm excitation and a Fourier transform spectrometer. These spectra clearly indicate that the chromophore modes are observable over those of the protein with no signs of interference below 1800 cm-1. The chromophore modes are dominated by those of the bacteriochlorophylls (BChl a), and it is estimated that, in the P state, ca. 65% of the Raman intensity of the BChl a modes arises from the primary donor. This permits the direct observation of a vibrational spectrum of the primary donor at preresonance with the excitonic 865-nm band. The Raman spectrum of oxidized reaction centers in the presence of ferricyanide clearly exhibits bands arising from a BChl a+ species. The magnitude of the frequency shift of a keto carbonyl of neutral P from 1691 to 1717 cm-1 upon P+ formation strongly suggests that one BChl molecule in P+ carries nearly the full +1 charge. Our results indicate that the unpaired electron in P.+ does not share a molecular orbital common to the two components of the dimer on the time scale of the resonance Raman effect (ca. 10(-13) s).     

毛姜花油细胞原位拉曼光谱研究

为避免复杂的样品的制备及提取过程,最大限度避免精油活性成分变化,常温下,用拉曼光谱原位分析毛姜花油细胞中精油。样品切片后置于共聚焦显微拉曼光谱仪下,用10倍物镜可观察到油细胞。油细胞精油的拉曼光谱与1,8-桉油精拉曼光谱非常相似。以毛姜花油细胞/1,8-桉油精的拉曼峰为序,较强峰出现在2928/2 921、647/652 cm~(-1),次强峰出现在540/545、808/813、915/920、926/930、1 012/1 016、1 075/1 080、1 270/1273、1 427/1 432 cm~(-1)。在油细胞中出现的强峰、次强峰与1,8-桉油精的拉曼峰一致,说明毛姜花油细胞中油的主要成分为1,8-桉油精。毛姜花油细胞的25条拉曼峰都与1,8-桉油精的拉曼峰有很好的对应关系。