Macromolecular biophysics of the plant cell wall: Concepts and methodology

Plant cell walls provide form and mechanical strength to the living plant, but the relationship between their complex architecture and their remarkable ability to withstand external stress is not well understood. Primary cell walls are adapted to withstand tensile stresses while secondary cell walls also need to withstand compressive stresses. Therefore, while primary cell walls can with advantage be flexible and elastic, secondary cell walls must be rigid to avoid buckling under compressive loads. In addition, primary cell walls must be capable of growth and are subjected to cell separation forces at the cell corners. To understand how these stresses are resisted by cell walls, it will be necessary to find out how the walls deform internally under load, and how rigid are specific constituents of each type of cell wall. The most promising spectroscopic techniques for this purpose are solid-state nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR) and Raman microscopy. By NMR relaxation experiments, it is possible to probe thermal motion in each cell-wall component. Novel adaptations of FTIR and Raman spectroscopy promise to allow mechanical stress and strain upon specific polymers to be examined in situ within the cell wall.     

Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.     

Maturation of 5-S rRNA: ribonuclease E cleavages and their dependence on precursor sequences

By measuring the rhodopsin--bathorhodopsin, isorhodopsin--bathorhodopsin, rhodopsin--isorhodopsin and rhodopsin--meta-II difference spectra with the method of Fourier-transform infrared spectroscopy we have identified the C = N stretching vibration of the protonated retinylidene Schiff base of rhodopsin, isorhodopsin and bathorhodopsin. In contrast to resonance Raman spectroscopy additional strong bands were observed between 1700 cm-1 and 1620 cm-1. Most of them depend on the isomeric state of the chromophore. The origin of these bands will be discussed. In the fingerprint region isorhodopsin and bathorhodopsin are quite similar but no similarities with infrared spectra of model compounds of any isomeric composition are observed. Therefore, no conclusions on the isomeric state of the retinal in bathorhodopsin can be drawn. We provide evidence for the modification of one or two carboxylic group(s) during the rhodopsin--bathorhodopsin and isorhodopsin--bathorhodopsin transition.     

A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin

A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.     

大鼠大脑皮质与纹状体显微拉曼光谱的研究

用Spex-1428显微激光拉曼光谱仪测定正常大鼠大脑皮质和纹状体的激光拉曼光谱的变化,发现正常大鼠大脑皮质和纹状体的激光拉曼光谱在模式上大同小异,包含有十分丰富的生物大分子结构信息。结果如下：（1）在大脑皮质和纹状体同时出现且性状亦相似的有以下一些特征峰：808cm^-1的特征峰,对应于A型DNA的特征峰;832cm^-1和836cm^-1对就于酪氨酸（Tyr)环的振动峰;1020cm^-1和1046cm^-1相当于蛋白质中氨基酸内的C－N伸缩振动峰;1330cm^-1相当于腺嘌呤环的C＝C和C－N伸缩振动峰;1544cm^-1相当于酰胺Ⅱ的N－H平面内弯曲振动和C－N伸缩援峰;1684cm^-1对应蛋白质二级结构中的转角（turn)结构。（2）大脑皮质较纹状体明显的特征峰：1092cm^-1的DNA骨架的对3称振动峰;1350cm^-1的色氨酸峰;1690cm^-1为尿嘧啶的C＝O振动峰。（3）纹状体较大脑皮质明显的特征峰;1450－1463cm^-1为蛋白质CH2弯曲振动的特征峰带,纹状体在此区段有1454cm^-1峰,而大脑皮质在此区域比较低平;1490cm^-1,为鸟嘌呤的特征峰。结果显示：显微拉曼光谱揭示的大脑皮质和纹状体的生物大分子的结构成分信息十分丰富,既有共性也有差异,显微拉曼光谱是一种十分灵敏的研究手段。     

Effect of selective growth media on the differentiation of Salmonella enterica serovars by Fourier-Transform Mid-Infrared Spectroscopy

Salmonella enterica serovars are prevalent foodborne pathogens responsible for high numbers of salmonellosis each year. Complex Fourier-transform infrared (FTIR) spectra offer unique biochemical fingerprints of bacteria with bands due to major cellular components. Growth media effects on discrimination of Salmonella serovars by FTIR spectroscopy were investigated and a novel sample preparation technique was developed. S. enterica strains from six serovars were grown on xylose lysine desoxycholate (XLD), Miller-Mallinson (MM), and plate count (PCA) agar as a control (37 degrees C, 24 h). Isolated colonies were suspended in 50% acetonitrile and centrifuged; the remaining pellet was placed on an AMTIR (attenuated total reflectance) crystal and dried under vacuum. Classification models (Soft Independent Modeling of Class Analogy, SIMCA), generated from derivatized infrared spectra (1300-900 cm-1 or 1200-900 cm-1), successfully discriminated among Salmonella strains with major discrimination from 1000-970 cm-1 associated to stretching modes of O-specific polysaccharide chains of lipopolysaccharides. Sample treatment with acetonitrile enhanced safe handling of the bacteria, removed interfering signals and improved the discriminating ability of SIMCA. All media were able to discriminate the S. enterica strains studied, varying in discriminating peaks and class distances in SIMCA classification. This methodology, with the production of large libraries of pathogenic bacteria, could be applied for the rapid monitoring of bacterial contamination in food with minimal sample manipulation.     

Changes in Dehydrodiferulic Acids and Peroxidase Activity against Ferulic Acid Associated with Cell Walls during Growth of Pinus pinaster Hypocotyl

Hydroxycinnamic acids associated with hypocotyl cell walls of dark-grown seedlings of Pinus pinaster Aiton were extracted with 1 N NaOH and identified by gas chromatography-mass spectrometry. The main hydroxycinnamic acid found was ferulic acid. Diferulic acid dehydrodimers were also found, with the 8,8-coupled isomer (compound 11) being the dehydrodiferulate present in the highest amount. However, the 5,5-coupled isomer, commonly referred to referred to as diferulic acid, was not detected. Two truxillic acids, 4-4[prime]-dihydroxy-3-3[prime]-dimethoxy-[alpha]-truxillic acids I and II, were tentatively identified. The 8,8-coupled dehydrodiferulic acid (compound 11) was the phenolic acid that showed the most conspicuous changes with hypocotyl age as well as along the hypocotyl axis. Peroxidase activity against ferulic acid was found in the apoplastic fluid as well as being ionically and covalently bound to the cell walls. The peroxidase activity increased with hypocotyl age as well as from the subapical toward the basal region of the hypocotyls. A key role in the cell-wall stiffening of 8,8 but not 5,5 dimerization of ferulic acid catalyzed by cell-wall peroxidases is proposed.     

Interpretation of DNA vibration modes. II--The adenosine and thymidine residues involved in oligonucleotides and polynucleotides

Normal coordinate analysis of the adenosine and thymidine residues involved in the right- and left-handed conformations of oligonucleotides and polynucleotides has been performed. The valence force field, employed in this work, allowed recently to reproduce the vibrational spectra of 2'-deoxythymidine and 2'-deoxyadenosine. The calculated wavenumbers based on a non-redundant set of internal coordinates have been compared to the Raman and infrared peak positions arising from A, B, C, D and Z conformations, in the 1550-1250 cm-1 and 800-600 cm-1 spectral regions: i.e. characteristic of adenosine and thymidine residues. Moreover, a systematic study has been performed on the evolution of the vibrational wavenumbers as a function of the glycosidic angle (chi) and the sugar pucker conformation.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Characterization of the different peripheral light-harvesting complexes from high- and low-light grown cells from Rhodopseudomonas palustris

In this paper we demonstrate that the spectroscopically different peripheral light-harvesting complexes from Rhodopseudomonas palustris, strain 2.6.1, isolated from high- and low-light grown cells have widely differing bacteriochlorophyll a (BChl a) resonance Raman spectra in the high-frequency carbonyl region (1550-1750 cm-1). Complexes synthesized in low-light grown cells exhibit Raman spectra characteristic of B800-850 and B800-820 complexes, depending on the excitation conditions. The in vivo strategy for low-light adaptation in this bacterium is thus somewhat different from that generally encountered in the Rhodospirillaceae. In these bacteria, as typified by Rps. acidophila and Rps. cryptolactis, low-light conditions induce the synthesis of B800-820 only complexes in which the hydrogen bonds between the acetyl carbonyl and the B850 binding pocket are broken, inducing changes in the absorption properties of the monomeric bacteriochlorophylls. In the case of Rps. palustris, additional spectral effects occur due to the coupling of the electronic levels of the differently interacting dimers. The extensive use of differential alpha/beta-polypeptide expression [Tadros et al. (1993) Eur. J. Biochem. 217, 867-875] thus allows Rps. palustris to alter its BChl a binding site environments causing the observed spread of BChl a Qy transitions, ranging from 801 to 856 nm.     

Alterations in Structural Polysaccharides during Liquefaction of Tomato Locule Tissue

The locule tissue of tomato (Lycopersicon esculentum, Mill.) undergoes extensive liquefaction during ripening. In this study, the solubility, molecular mass, and glycosyl composition of locule pectic and alkali-soluble polysaccharides were examined with the aim of identifying features contributing to the unique properties of this tissue. Ethanol-insoluble solids were prepared from de-seeded locule tissue from tomato fruit at the immature green (IMG), mature green, and breaker stages of development. Ethanol-insoluble pectins were extracted sequentially in H2O, 50 mM trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, 50 mM Na2CO3, and 4 M KOH. At the IMG stage, nearly 85% of the locule pectins were solubilized by water, trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, and Na2CO3 solutions. Solubility increased only slightly with further locule development. The noncovalently associated polymers were of high molecular mass throughout liquefaction. Polymers extracted in mild alkali were of considerably lower molecular mass. Locule pectins in IMG fruit were heavily glycosylated with galactose, arabinose, and xylose. All pectin classes exhibited similar deglycosylation trends during liquefaction. Locule hemicelluloses were rich in glucose, xylose, and arabinose. These polymers collectively showed molecular mass downshifts with minimal compositional changes during liquefaction. The KOH-soluble material also included xylose-rich acidic polymers not matching the neutral sugar profile of the noncovalently associated pectic polymers.     

Raman, infrared, and circular dichroism spectroscopic studies on metallothionein: a predominantly "turn"-containing protein

Raman and IR spectra of rabbit liver metallothionein 1 (MT-1) containing 7 mol of either cadmium or zinc ions reveal high-lying amide III bands between 1290 and 1330 cm-1, indicative of beta-turns. A comparison of the splitting pattern in the amide III region below 1290 cm-1 and in the amide I band between 1600 and 1700 cm-1, with the normal-mode calculations of Lagant et al. [Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984a) Eur. J. Biochem. 139, 137-148; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984b) Eur. J. Biochem. 139, 149-154; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984c) J. Raman Spectrosc. 15, 421-423] and Krimm and Bandekar [Krimm, S., & Bandekar, J. (1980) Biopolymers 19, 1-29], suggests that metal-bound (holo) MT-1 consists largely of beta-turns of type II. In contrast, the metal-free (apo) protein displays a predominantly unordered conformation. The Raman spectra of the holoproteins below 1000 cm-1 are characterized by several unusual skeletal stretching and bending modes. The spectral pattern between 760 and 800 cm-1 in conjunction with the splitting of the amide I band agrees closely with the normal-mode calculations of Lagant et al. (1984b) on model peptides and is indicative of the presence of type III beta-turns (or 3(10)-helical segments) in MT-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flavins of NADPH-cytochrome P-450 reductase: evidence for structural alteration of flavins in their one-electron-reduced semiquinone states from resonance Raman spectroscopy

The mechanism of electron transfer from NADPH to cytochrome P-450 through FAD and FMN of the reductase is largely unknown. In this paper, we report the resonance Raman spectral properties of the oxidized and the semiquinonoid states of the flavins in the holoenzyme and the FMN-depleted forms, respectively, of detergent-solubilized rabbit liver microsomal NADPH-cytochrome P-450 reductase. The resonance Raman spectra of the oxidized forms [FAD; FMN] and [FAD;-] were essentially identical, indicating similar binding interactions of these flavins with the protein. To the contrary, the spectra of the semiquinonoid FADH. and FMNH. forms revealed significant spectral differences. Both O2-unstable species, characterized as [FADH.; FMNH2] and [FADH.;-] excited at 568.2 nm, have dominant spectral peaks at approximately 1611, 1539-1543, 1377, 1305, 1263, and 1226 cm-1. However, in the O2-stable [FAD; FMNH.] species, resonance Raman bands were located at 1611, 1532, 1388, 1304, 1268, and 1227 cm-1 when excited at the same wavelength. The approximately 10-cm-1 shifts of the 1532- and 1388-cm-1 bands suggest that the environments surrounding rings II and III of the isoalloxazines change upon reduction to semiquinonoid forms. It is proposed that N1 of FADH. (as a hydrogen-bond acceptor) and N5 of FMNH. (as donor) provide the distinguishing flavin-protein interactions in the semiquinonoid states. Furthermore, the resonance Raman spectra of the semiquinonoid species appear to be missing a number of bands assigned to ring I vibrations in the spectra of the oxidized flavins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nature of the IR spectra of intact cells of Gram-negative microorganisms

The character of changes in the infrared spectra of E. coli in the process of their mechanical disintegration has been studied. The destruction of E. coli cell structures has been shown to produce no changes in the optical density of the main analytical absorption bands in infrared spectra. This fact suggests that the infrared absorption spectra of E. coli are the sum of the spectra of all chemical components of the cell, which is confirmed by the infrared spectral study of E. coli cell-wall preparations. Similar results have been obtained in the study of the preparations of B. pertussis cell walls, protoplasts and intact cells.     

Polarized infrared microspectroscopy of single spruce fibers: hydrogen bonding in wood polymers

We studied wood polymers in their native composite structure using mechanically isolated single spruce (Picea abies [L.] Karst.) fibers. Dichroic infrared spectra of fibers placed in a custom-built microfluidic cuvette were acquired in air, in liquid (heavy) water, and in liquid dimethylacetamide using a novel combination of synchrotron-based Fourier transform infrared microspectroscopy with polarization modulation. Differences were observed in the O-H stretching frequency region of the spruce spectra upon changing the ambient conditions. Analysis of these spectral variations provides information on hydrogen bonding, orientation, and accessibility of structural units of the wood polymers in the spruce cell walls. Our in situ approach contributes to a further understanding of the structural details of wood polymers in their native setting.     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox-linked conformational changes in proteins detected by a combination of infrared spectroscopy and protein electrochemistry. Evaluation of the technique with cytochrome c

We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c, because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reduced-minus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-1 and 1800 cm-1.     

Characterization of cross-linking of cell walls of Bacillus subtilis by a combination of magic-angle spinning NMR and gas chromatography-mass spectrometry of both intact and hydrolyzed 13C- and 15N-labeled cell-wall peptidoglycan.

Cross-polarization magic-angle spinning 13C and 15N NMR, rotational-echo double resonance 13C NMR, and delays alternating with nutation for tailored excitation-difference 13C NMR spectra have been obtained from lyophilized cell walls of Bacillus subtilis grown on a synthetic medium containing D,L-[2-13C, 15N]aspartate and D-[1-13C]alanine. Label from aspartate is incorporated into D-glutamic acid and m-diaminopimelic acid of cell-wall peptidoglycan, while label from alanine appears in the C-1 positions of both D- and L-alanyl residues. The cross-link index (the fraction of peptide stems joined by an isopeptide covalent bond) is obtained directly from analysis of the results of the 13C NMR experiments. However, specific isotopic enrichments of cell-wall components cannot be obtained from NMR data alone. The latter are determined either from a gas chromatographic-mass spectrometric analysis of the amino acids derived from hydrolysis of cell-wall peptidoglycan, or from a combination of NMR and gas chromatographic-mass spectrometric results. The combined analysis is overdetermined and so involves the least error for evaluations of both the cross-link index and the isotopic enrichments. The cross-link index is 0.33 +/- 0.03 for cell walls of B. subtilis grown in the presence of the antibiotic, cephalothin.     

Fourier-transform,infrared difference-spectrometry for structural analysis of dextrans

The Fourier-transform (F.t.), infrared (i.r.) spectra of a series of branched dextrans were examined. The dextrans studied were those from the N R R L collection designated Leuconostoc mesenteroides B-1142, B-1191, B-1299 fraction S, B-1355 fraction S, B-1402, and B-1422, and Streptobacterium dextranicum B-1254 fractions S[L] and L[S]. The spectrum of a levan, N R R L L. mesenteroides B-523 fraction M, was also examined, for comparison with the spectra of the dextrans. Meaningful results were obtained by “weight-normalizing” the spectral absorbance to that of the dextran of very low degree of branching (dextran B-1254 fraction L[S]), and then subtracting this spectrum of linear dextran from each of the other polysaccharide spectra. The resulting i.r.-absorbance difference-spectra were plotted, at uniform scale-expansion across the 1800-400-cm^?1 region, resulting in difference-absorbance features at ≈ 1100 and ≈ 800 cm^?1 for all branched dextrans. These absorbance differences could be correlated to the type and degree of dextran branching, which had previously been established by permethylation analysis. It was concluded that such F.t.-i.r. difference-spectra have general application for the structural analysis of polysaccharides.     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.