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.     

Raman intensities of carbon-carbon stretching modes in a model membrane

Laser-Raman spectra of L-α-dimyristoylphosphatidylcholine (DMPC) liposomes in the spectral range 1000–1200 cm^?1 were obtained as a function of temperature from ?80 to +50°C. The triplet found in this spectral region was resolved into Lorentzian components by means of an iterative computer program. The peak intensities, band widths, and band areas of the resolved 1062 cm^?1 and 1130 cm^?1 bands, assigned to CC stretching vibrations of trans segments, were evaluated as a function of temperature. While the peak intensities of the bands decrease substantially with temperature, the band widths show a considerable increase. The change in band areas is therefore smaller than the change in peak heights. Experiments with all trans carboxylic acids showed that in these compounds the area of the Raman bands at 1062 cm^?1 and 1130 cm^?1 is proportional to the number of trans bonds. The variation with temperature of the number of trans and gauche bonds in the studied phospholipid is reflected by the change of the area of the 1130 cm^?1 Raman band.     

Performance of a time-resolved IR facility for assessment of protonation states and polarity changes in carboxyl groups in a large membrane protein,mammalian cytochrome c oxidase,under turnover conditions in a sub-millisecond time resolution

Time-resolved IR analyses for the protonation and polarity changes of carboxyl groups involved in proton pump enzymes under turnover conditions are indispensable for elucidation of their proton-pump mechanisms. We have developed a new time-resolved infrared facility by introducing a flow system for transferring highly concentrated and thus viscous protein solution to a thin (50?μm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350?cm^?1 as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O₂ supply system enables the sub-millisecond time scale infrared measurements of the O₂ reduction coupled with proton pumping by bovine cytochrome c oxidase (CcO) initiated by CO-flash photolysis in the COOH (1725–1770?cm^?1) region with the accuracy of about 10?μO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744?cm^?1 assignable to protonation of a carboxyl group coupled with a single electron transfer to the O₂ reduction center within 1?ms after initiation of the reaction. The results suggest that the facility detects protonation of a single carboxyl group included in large proteins like as CcO (210?kDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750?cm^?1 and 1760?cm^?1, without significant intensity changes. These findings show the performance of this facility sufficiently high for providing crucial information for understanding the proton transferring mechanisms of protein carboxyl groups.     

Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm^?1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the C? H stretching vibrational population between 2,984 and 2,780 cm^?1. Carbohydrate content was determined using integration over a C? O and C? O? C stretching band area between 1,180 and 1,133 cm^?1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129. © 2008 Wiley Periodicals, Inc.     

Etude par Spectroscopie Infrarouge de la Transformation Hélice—Chaine Statistique des Polypeptides. I. Etude Comparée de l'Interaction de la Poly-L-alanine et d'un Amide Modèle avec l'Acide Trifluoroacétique

Infrared spectra of poly-L -alanine in trifluoroacetic acid-chloroform mixtures have been investigated and compared with those of a model amide (N-methylacetamide). The purpose of this work is to determine the nature of peptide-acid specific interactions responsible for the helix-random coil transition of polymer chains. Analysis is made in using amide (A, I, II, III) and acid (νC?O, νOH) vibrations which are specially sensitive to molecular interactions. We examined a model compound to determine the spectral characteristics of the different complexes or species formed between amide and acid. At a low acid concentration, hydrogen-bonded complexes: ? (NH) C?O…?HOOCCF₃ (1) are evidenced but no association between amide NH and acid CO groups (complexes A) is observed. For higher acid concentrations complexes (I) are progressively changed into ions pairs and free ions, which result from amide protonation by acid, according to the exothermic equilibrium (I)?? (NH)COH⁺, ^?OOCCF₃(II). Amidium and carboxylate bands are localized between 1680–1705 cm^?1 and 1620–1625 cm^?1, respectively. If the cation band is always clearly seen, the anion band is only observed for the most acidic solutions. For the polymer, a gradual complexation of type (I) is observed for all acid concentrations. From our results, the assumption of an (A) type interaction seems very unlikely but cannot be excluded. Moreover, proton transfer—similar to that observed with a model amide—is never evidenced since, in particular, the amidium band characteristic of protonation is never seen. In contrast to previous investigations, we conclude that the helix-random coil transition of polypeptides is not due to the protonation of the peptide functions. This transition does suggest a strong interaction by hydrogen bonds between polymer and acid molecules.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

Aerobic biodegradation of Azo dye by Bacillus cohnii MTCC 3616; an obligately alkaliphilic bacterium and toxicity evaluation of metabolites by different bioassay systems

An obligate alkaliphilic bacterium Bacillus cohnii MTCC 3616 aerobically decolorized a textile azo dye Direct Red-22 (5,000 mg?l^?1) with 95 % efficiency at 37 °C and pH?9 in 4 h under static conditions. The decolorization of Direct Red-22 (DR-22) was possible through a broad pH (7–11), temperature (10–45 °C), salinity (1–7 %), and dye concentration (5–10 g?l^?1) range. Decolorization of dye was assessed by UV–vis spectrophotometer with reduction of peak intensity at 549 nm (λ _max). Biodegradation of dye was analyzed by Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). The FTIR spectrum revealed that B. cohnii specifically targeted azo bond (N=N) at 1,614.42 cm^?1 to break down Direct Red-22. Formation of metabolites with different retention times in HPLC analysis further confirmed the degradation of dye. The phytotoxicity test with 5,000 mg?l^?1 of untreated dye showed 80 % germination inhibition in Vigna mungo, 70 % in Sorghum bicolor and 80 % in Vigna radiata. No germination inhibition was noticed in all three plants by DR-22 metabolites at 5,000 mg?l^?1. Biotoxicity test with Artemia salina proved the lethality of the azo dye at LC₅₀ of 4 and 8 % for degraded metabolites by causing death of its nauplii compared to its less toxic-degraded metabolites. Bioaccumulation of dye was observed in the mid-gut of A. salina. The cytogenotoxicity assay on the meristematic root tip cells of Allium cepa further confirmed the cytotoxic nature of azo dye (DR-22) with decrease in mitotic index (0.5 % at 500 ppm) and increase in aberrant index (4.56 %) over 4-h exposure period. Genotoxic damages (lagging chromosome, metaphase cluster, chromosome bridges, and dye accumulation in cytoplasm) were noticed at different stages of cell cycle. The degraded metabolites had negligible cytotoxic and genotoxic effects.     

Characterization of hydroxyl groups of highly crystalline β-chitin under static tension detected by FT-IR

The different behavior of the OH groups of a highly crystalline β-chitin from Lamellibrachia sp. was revealed by FT-IR spectroscopy with static tension applied in the direction of the chain. Both OH groups shifted to higher wavenumbers under the tension, possibly because the force constant of the OH vibration intensified due to the erosion of hydrogen bonds, but the shift in the OH peak occurring at 3435 cm⁻¹ was 14% greater than that of the peak at 3470 cm⁻¹. The band at 3435 cm⁻¹ was assigned to O(3)H and that at 3470 cm⁻¹ to O(6)H, because the intramolecular hydrogen bond of O(3)H?O(5) occurs along the chitin chain's backbone and is likely to be more affected under tensile stress as a load carrier, than O(6)H?O(7)C, which is an intermolecular hydrogen bond located in a branch of the chain. Computation of an IR spectrum under stretching was conducted and supported the assignment of the two OH groups.     

Conformational dependence of the Raman scattering intensities from polynucleotides. 3. Order-disorder changes in helical structures

Raman spectra are presented on ordered and presumably helical structures of DNA and RNA as well as the poly A·poly U helical complex, polydAT, and the helical aggregates of 5′-GMP and 3′-GMP. The changes in the frequency and the intensity of the Raman bands as these structures undergo order-disorder transitions have been measured. In general the changes we have found can be placed into three categories: (1) A reduction in the intensities of certain ring vibrations of the polynucleotide bases is observed when stacking or ordering occurs (Raman hypochromism). Since the ring vibrational frequencies are different for each type of base, we have been able to obtain some estimate of average amount of order of each type of base in partially ordered helical systems. (2) A very large increase in the intensity of a sharp, strongly polarized band at about 815 cm^?1 is observed when polyriboA and polyriboU are formed into a helical complex. Although this band is not present in the separated chains at high temperature, a broad diffuse band at about 800 cm^?1 is present. The 815 cm^?1 band undoubtedly arises from the vibrations of the phosphate-sugar portions of the molecule and provides a sensitive handle to the back-bone conformation of the polymer. This band also appears upon ordering of RNA, formation of the helical aggregate of 5′-riboGMP, and to some extent in the selfstacking of the polyribonucleotides polyA, polyU in the presence of Mg⁺⁺, PolyC, and polyG. No such intense, polarized band is found, however, in ordered DNA, polydAT, or the 3′-riboGMP aggregate, although there is a conformationally independent band at about 795 cm^?1 in DNA and polydAT. (3) Numerous frequency changes occur during Conformational changes. In particular the 1600–1700 cm^?1 region in D₂O shows significant conformationally dependent changes in the C?O stretching region analogous to the changes in this region which have been observed in these substances in the infrared. Thus, Raman scattering appears to provide a technique for simultaneously observing the effects of base stacking, backbone conformation and carbonyl hydrogen bonding in nucleic acids in moderately dilute (10–25 mg/ml) aqueous solutions.     

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2?cm^?1, that are about 40% smaller than the D-value of the wild-type protein. The ⁵⁷Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01?mm/s at 4.2?K rather than δ=0.32?mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79?mm/s at 4.2?K, consistent with tetrahedral Fe²⁺(Cys)₃(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=?7±1?cm^?1, E/D=0.09 for form A and D=+6.2±1.3?cm^?1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g _z =2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77?K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe²⁺ site. Studies in different buffers in the pH?6–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700?cm^?1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300?cm^?1; a third d-d transition, if present, must occur with energy lower than 3300?cm^?1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100?cm^?1 in form A and 5350, 6380?cm^?1 in form B.     

Infrared-spectral characteristics of some acetylated,anomeric glycosides

Barker and co-workers had described the C-1-H deformation bands in the ranges 844 ±8 cm^?1 and 891 ±7 cm^?1 as characteristic bands for the α and β anomers, respectively, of hexo- and pento-pyranoses and -pyranosides, and their derivatives. Later, Audichya and co-workers reported the presence of the 844 ±8-cm^?1 band for both anomers of some aryl d-glucoside derivatives, making the applicability of the earlier findings doubtful. Examination by us of the i.r. spectra of some aryl glycoside derivatives suggested that the origin of the band at 844 ±8 cm^?1 for the β anomers of the p-substituted-aryl glycoside derivatives studied by Audichya et al. could be a CH, out-of-plane deformation-mode of the substituted aromatic ring. Also, their further claim of a characteristic band in the region 961-957 cm^?1 for α anomers is shown to be of little diagnostic value. The relative intensities of bands in the COC stretching region, 1100-1000 cm^?1, and a band near 300 cm^?1 in the COC deformation region, found only for the β anomers, are shown to be helpful in differentiating the anomers of some peracetylated alkyl and aryl glycosides.     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm^?1 shift from 1306 cm^?1 in aqueous solution to 1314 cm^?1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding ability. In non-hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm^?1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm^?1; in water, a strong hydrogen-bonding solvent, 1306 cm^?1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm^?1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

RNA Binding Efficacy of Theophylline,Theobromine and Caffeine

Abstract

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 ± 5%), whereas moderate and comparatively less binding activity for theobromine (45 ± 5%) and caffeine (30 ± 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm^?1), theobromine (3379.8 cm^{? 1}) and caffeine (3343 cm^?1) as compared to the free RNA (3341.6 cm^?1). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (υC=O) of both drug (υC=O=1718, 1666 cm^?1) as well as RNA (υC=O=1699, 1658 cm^?1) disappeared and a new vibration band appeared around 1703 cm^?1, indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theo- bromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.     

Conformational geometry and vibrational frequencies of nucleic acid chains.

Normal coordinate analysis of diethyl phosphate has been made, which predicts all observed Raman frequencies in the range 170–1300 cm^?1. The force constants from this calculation have been transferred to a vibrational calculation for a simplified model of the backbone of nucleic acids, which also involves the ? O? PO₂^?? O phosphate group and the ? C₅′? C₄′? C₃′? linkage of the ribose. The coordinates of these atoms are those recently given by Arnott and Hukins, which place the ribose ring of B-DNA in a C₃′-exo conformation. This simple polymer model appears to be able to describe adequately the frequency-dependent changes observed in the Raman spectra arising from the backbone vibrations of nucleic acid in going from the B- to A-form. The symmetric ? O? P? O? diester stretch increases in frequency from about 787 cm^?1 in the B-form to 807 cm^?1 in the A-form. The increased frequency characteristic of the A-form is due to the combining of the diester stretch with vibrations involving the C₅′, C₄′, and C₃′ nuclei. The frequency of the symmetric ? O? P? O? diester stretch is shown to be very dependent on the conformation of the ribose ring, indicating that in polynucleotides the ribose ring takes on one of two rigid conformations: C₃′-endo for A-form or C₃′-exo for B-form and “disordered” polynucleotides. The calculation lends confirmation to the atomic coordinates of Arnott and Hukins since the use of other geometries with the same force constants failed to give results in agreement with experimental evidence. The calculations also demonstrate the lowering effect of hydration on the anionic PO stretching frequencies. Experimental results show that the 814-cm^?1 band observed in the spectra of 5′GMP gel arises from a different vibrational mode than that of the 814-cm^?1 band of A-DNA.     

Graphite oxide‐dispersed CdTe quantum dots nanocomposite for flexible display luminescent membranes

A quantum dot (QD) dispersant material was prepared using graphite oxide (GO). Luminescent films were prepared using polyvinyl alcohol as the polymer matrix. First, water‐soluble CdTe QDs were prepared by wet chemistry and GO was synthesized using a modified Hummers method. X‐Ray diffraction tests showed that the GO reflection peak [001] was 11.9°, which indicates that the d‐spacing is 0.7431 nm; atomic force microscopy showed a GO thickness of 200 nm. Fourier transform infrared spectra showed vibrations at 1624 cm^?1 for the carbonyl groups, and 3260 cm^?1 for the GO samples; the ‐C–O vibration was at 1320 cm^?1 and ‐COOH, ?OH vibrations were at 950 cm^?1. Fluorescent tests showed that pH had an impact on the QD colloidal stability. GO was neutralized before use as the host media for the GO/QDs nanocomposite. The results proved that the resultant nanocomposite is promising for use in brightness enhancement films in flexible displays.     

Fourier-transform infrared spectroscopy of Pediastrum duplex: characterization of a micro-population isolated from a eutrophic lake

Fourier-transform infrared (FTIR) spectroscopy was carried out on single colonies of Pediastrum duplex present in air-dried preparations of mixed phytoplankton samples isolated from a eutrophic freshwater lake. FTIR absorption spectra had 12 distinct bands over the wavenumber range 3300–900?cm^?1 which were tentatively assigned to a range of chemical groups, including –OH (residual water, wavenumber 3299?cm^?1), –CH2 (lipid, 2924), –C=O (cellulose, 1739), amide (protein, 1650 and 1542), >P=O (nucleic acid, 1077) and –C–O (starch, 1151 and 1077). Measurement of band areas identified residual water, protein and starch as the major detectable constituents. Areas of single bands and combined bands of –CH₂, –C–O and >P=O species normalized to protein (to correct for differences in specimen hydration and thickness) showed wide variation between colonies, indicating environmental heterogeneity. Correlation analysis demonstrated close statistical associations between different molecular species. Particularly high levels of correlation between bands 3/4 (CH₂), 6/7 (amide) and 8/9 (–CH₃) was consistent with their joint origin from the same molecular species. The isolation of bands 11 and 12 in the correlation pattern was confirmed by factor analysis, suggesting that variation in the level of starch is statistically unrelated to other macromolecules being monitored. The use of FTIR spectroscopy to characterize an algal micro-population within mixed phytoplankton has potential for future studies on biodiversity and environmental interactions at the species level.     

Structural analysis of some soluble elastins by means of FT‐IR and 2D IR correlation spectroscopy

Fourier transform infrared (FT‐IR) spectroscopy combined with 2D correlation spectroscopy has been used to offer some information about stability and structure of some soluble elastins. Temperature has been chosen as the perturbation to monitor the infrared behavior of various soluble elastins, namely, α‐elastin p, α‐elastin, and k‐elastin. In the 3800–2700 cm^?1 region, the H‐containing groups were analyzed. The bonded hydroxyls are found to decrease prior to the NH‐related hydrogen bonds and also to the conformational reorganization of hydrocarbon chains. The transition temperatures were evaluated and they were found to agree with those obtained from DSC data. The FTIR spectra and their 2nd derivatives denote that α‐ elastins exhibited amide‐I, ‐II and ‐III bands at 1656, 1539 and 1236 cm^?1, respectively, while in k‐elastin these bands were found at 1652 cm^?1 for amide I, 1540 cm^?1 for amide II and 1248 cm^?1 for amide III. The macroscopic IR finger‐print method, which combines: general IR spectra, secondary derivative spectra, and 2D‐IR correlation spectra, is useful to discriminate different elastins. Thus using the differences of the position and intensity of the bands from “fingerprint region” of studied elastins, which include the peaks assigned to C?O, C? C groups from α‐helix, β‐turn, and the peaks assigned to the amide groups, it is possible to identify and discriminate elastins from each others. Furthermore, the pattern of 2D‐IR correlation spectra under thermal perturbation, allow their direct identification and discrimination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1072–1084, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ～ 1650 cm^?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm^?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm^?1 component in the native collagen spectrum diminishes and the 1633-cm^?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm^?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.     

Infrared absorption and raman scattering of sulfate groups of heparin and related glycosaminoglycans in aqueous solution

The i.r. spectra for aqueous solutions of sulfated glycosaminoglycans and model compounds in the transmittance “window” region of the solvent (1400-950 cm^?1) are dominated by the strong and complex absorption centered at ～1230 cm^?1 and associated with the antisymmetric stretching vibrations of the SO groups. Primary and secondary O-sulfate groups absorb at somewhat higher frequencies (1260-1200 cm^?1) than N-sulfates (～1185 cm^?1). Each sulfate band lends itself to quantitative applications, especially within a given class of sulfated polysaccharide. Laser-Raman spectra of heparin and model compounds have been obtained in aqueous solution and in the solid state. The most-prominent Raman peak (at ～1060 cm^?1) is attributable to the symmetrical vibration of the SO groups, with N-sulfates emitting at somewhat lower frequencies (～1040 cm^?1) than O-sulfates. The Raman pattern in the 950-800 cm^?1 region (currently used in the i.r. for distinguishing between types of sulfate groups) also involves vibrations that are not localized only in the COS bonds.