Raman spectroscopy and order in biological systems

The Raman spectra in the low 5–200 cm⁻¹ frequency region of metabolically activeE. coli cells have been analyzed to determine whether they are indicators of a possible in vivo underlying order by applying standard concepts derived from the Raman spectroscopy of crystalline systems with varying degrees of order. The analysis suggests that in-vivo space-time ordered structures involving amino acids associated with DNA exist since the low frequency lines of metabolically active cells can be assigned to lines seen in the spectra of crystals of given amino acids known to associated with DNA early in the lifetime of a cell.     

Subpicosecond dynamics in nucleotides measured by spontaneous Raman spectroscopy

The band widths in Raman spectra are sensitive to dynamics active on a time scale from 0.1 to 10 ps. The band widths of nucleotide vibrations and their dependence on temperature, concentration, and structure are reported. From the experimental band widths and second moments, it is derived that the adenine vibrations at 725, 1336, 1480, and 1575 cm⁻¹, and the uracil vibration at 787 cm⁻¹, are in the fast modulation limit. The correlation times of the perturbations are faster than 0.4 ps. Thermal melting of the helical structure in polynucleotides results in larger band widths, due to an increase in vibrational dephasing and energy relaxation as a consequence of the increased interaction of the base moieties with the solvent molecules. The band width of the 725 cm⁻¹ adenine vibration is dependent on the type and structure of the backbone. It is found to be perturbed by movements of the sugar-phosphate moiety relative to the base. The band width of the 1575 cm⁻¹ adenine vibration is found to be sensitive to the base-pairing interaction. From a comparison of the band widths in polynucleotides with a different base sequence (homopolymer vs alternating purine-pyrimidine sequence), it is concluded that resonant vibrational energy transfer between the base molecules is not important as a relaxation process for the vibrational band widths of nucleotides. Several theoretical models for the interpretation of band widths are discussed. The theory does not take into account the strong hydrogen-bonding nature water and hence fails to describe the observations in nucleotide-water systems. The bands of the carbonyl stretching vibrations are inhomogeneously broadened. The carbonyl groups have a strong dipolar interaction with the polar water molecules and are therefore strongly perturbed by coupling to the heatbath via hydrogen bonds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 751–763, 1997     

Structural analysis of arabinoxylans isolated from native and malted finger millet (Eleusine coracana, ragi)

Structural elucidation of purified arabinoxylans isolated from finger millet and its malt by methylation, GLC-MS, periodate oxidation, Smith degradation, NMR, IR, optical rotation, and oligosaccharide analysis indicated that the backbone was a 1,4-beta-D-xylan, with the majority of the residues substituted at C-3. The major oligosaccharide generated by endo xylanase treatment was homogeneous with a molecular weight of 1865 Da corresponding to 14 pentose residues as determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural analysis of this oligosaccharide showed that it contained 8 xylose and 6 arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3 (disubstituted).     

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K_d) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.     

The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy

The pH-dependent structure of calf thymus DNA is analyzed using Raman spectroscopy. The Raman spectra in the acidic region demonstrate that denaturation occurs in several steps. The binding of H⁺ to adenine and cytosine residues is accompanied by a decrease in the percentage of DNA in the B-conformation and a concurrent increase in a conformation most probably related to the C-form. The denaturation of DNA is observed at pH 3.3 and parallels the protonation of guanine bases. The Raman spectra of calf thymus DNA in the basic region (above pH 10) show that guanine residues are deprotonated at a lower pH value than are thymine residues. In addition, Raman spectra in the basic region detect conformational changes of the phosphate backbone different from those found in the acidic region.     

Study of ATP binding in the active site of Na,K-ATPase as probed by ultraviolet resonance Raman spectroscopy

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na⁺K⁺-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH₂, group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog β-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.     

Specificity of monoclonal antibodies generated against arabinoxylans of cereal grains

Xylooligosaccharides substituted by arabinose have been produced by degradation of wheat flour arabinoxylans with an endoxylanase. These oligosaccharides were coupled to carrier proteins (KLH and BSA) and three monoclonal antibodies were isolated. The specificity of antibody recognition was studied using arabino-xylo-oligosaccharides exhibiting different pattern of substitution by arabinose.

ELISA competition tests and molecular modelling suggest that the conformation adopted by beta-(1→4) linked xylose residues is an antigenic determinant recognized by the different antibodies. Arabinose was not specifically involved in the interaction of antibody and epitope.     

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.     

On the way to a quality control of the essential oil of fennel by means of Raman spectroscopy

Essential oils are one of the most valuable natural products. The price of special essential oils that can be purchased on the market strongly depends on the quality of the product. The quality, which depends on the quantitative and qualitative variation of different monoterpenes, varies with respect of the origin and the harvesting period. This contribution reports on a Raman spectroscopic study on the essential oil occurring in fennel. Cross-sections of fennel seed were investigated by use of Raman spectroscopy and Raman mapping to localize the essential oil and to analyze its chemical composition directly in the plant. Furthermore the practicability of a home-built mobile transportable Raman spectrometer to perform on-site measurements was successfully tested.     

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

The first investigation of the dynamics of a redox transition of an electron-transfer enzyme by time-resolved resonance Raman spectroscopy in combination with pulse-radiolytical reduction is described by an application to cytochrome c. A long-lived transient state is observed upon reduction of the alkaline form of cytochrome c as a distinct frequency shift of one resonance Raman band. From the frequency in the stable oxidized state, 1567 cm^?1, this particular resonance Raman band shifts within less than 1 μs to 1533 cm^?1 in the transient reduced state, which has a lifetime longer than 20 ms but shorter than a few seconds. Finally, in the stable reduced state, this band is located at 1547 cm^?1. According to a previous normal coordinate analysis, this resonance Raman band can be assigned predominantly to a stretching mode of the outermost C-C bonds in the four pyrrole rings of porphyrin. This vibrational mode is influenced by the protein most directly through the covalent thioether linkages of two cysteines to porphyrin. We interpret the long lifetime of the transient state as due to the slow return of Met-80 as sixth ligand to the heme iron upon reduction of the alkaline form of cytochrome c.     

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy

A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10^–10 M) solutions of doxorubicin or adriamycin (DOX), and 4O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.Abbreviations DNA deoxyribonucleic acid - DOX doxorubicin - SERS surface-enhanced Raman spectroscopy - THP-ADM 4O-tetrahydropyranyl adriamycin - PBS phosphate buffered saline Offprint requests to: M. Manfait     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.     

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.     

New enzyme-based method for analysis of water-soluble wheat arabinoxylans

Arabinoxylans (AX) are the predominant cell-wall polysaccharides in wheat flour. Water-extractable AX are essential for dough and bread properties and performance. However, there is no specific and accurate way of determining the content and structure of AX. An enzyme-assisted method employing an efficient enzyme mixture for the total hydrolysis of AX was developed in the present work. Enzymatic hydrolysis (EH) is a gentle method during which no unwanted sugar destruction occurs. Following EH, liberated monosaccharides were analysed by gas chromatography (GC) and liquid chromatography using HPAEC-PAD. The results were compared with acid methanolysis (AM) and acid hydrolysis (AH). EH performed better on commercially isolated AX samples than the reference method AM. Its action in the water extract from wheat flour was also more efficient than that of AM and comparable to the efficiency of AH. HPAEC-PAD revealed a significant amount of fructose in the water extract following EH, originating from fructans in wheat flour not detected in the GC analysis. The wheat flour examined contained 0.29% water-extractable AX. The arabinose/xylose ratio was 0.32. The enzyme-based method developed is applicable for comparison of different wheat flours and can be used in evaluating the effect of processing on the content and structure of water-extractable AX.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.