Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

Rapid discrimination of commercial strawberry cultivars using Fourier transform infrared spectroscopy data combined by multivariate analysis

To determine whether pattern recognition based on metabolite fingerprinting for whole cell extracts can be used to discriminate cultivars metabolically, leaves and fruits of five commercial strawberry cultivars were subjected to Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectral data from leaves were analyzed by principal component analysis (PCA) and Fisher’s linear discriminant function analysis. The dendrogram based on hierarchical clustering analysis of these spectral data separated the five commercial cultivars into two major groups with originality. The first group consisted of Korean cultivars including ‘Maehyang’, ‘Seolhyang’, and ‘Gumhyang’, whereas in the second group, ‘Ryukbo’ clustered with ‘Janghee’, both Japanese cultivars. The results from analysis of fruits were the same as of leaves. We therefore conclude that the hierarchical dendrogram based on PCA of FT-IR data from leaves represents the most probable chemotaxonomical relationship between cultivars, enabling discrimination of cultivars in a rapid and simple manner. The authors Suk Weon Kim and Sung Ran Min contributed equally to this work.     

Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ

A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.     

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.     

Fourier transform infrared (FTIR) spectroscopy for identifying Lactobacillus species

Abstract Fourier Transform infrared (FTIR) spectroscopy can be used to identify microorganisms. This study describes the influence of culture conditions on FTIR spectra and the discrimination of Lactobacillus species found in breweries. Fifty three Lactobacillus strains were analysed by FTIR spectroscopy and identification at the species level was correct for 94% of the strains, and at the strain level for 91% of the strains.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Rapid identification of coagulase-negative staphylococci by Fourier transform infrared spectroscopy

Coagulase-negative staphylococci (CNS), frequently associated with both community-acquired and nosocomial bloodstream infections, must be distinguished from Staphylococcus aureus for clinical purposes. Conventional methods are too laborious and time-consuming and often lack sensitivity to CNS. Fourier transform infrared (FTIR) spectroscopy combined with the use of a universal growth medium (Que-Bact Universal Medium No. 2) and chemometrics was evaluated for its potential as a rapid and simple clinical tool for making this distinction. FTIR spectra of 11 methicillin-sensitive and 11 methicillin-resistant CNS isolates as well as 25 methicillin-sensitive, 47 methicillin-resistant, 34 borderline oxacillin-resistant and 35 glycopeptide intermediate S. aureus isolates were obtained from dried films of stationary-phase cells grown on the universal medium. Principal component analysis (PCA), self-organizing maps, and the K-nearest neighbor algorithm were employed to cluster the different phenotypes based on similarity of their FTIR spectra. PCA of the first-derivative normalized spectral data from a single narrow region (2888-2868 cm(-1)) yielded complete differentiation of CNS from both methicillin-sensitive and methicillin-resistant S. aureus. The rate of correct classification was somewhat reduced, from 100% to 90%, after inclusion of borderline oxacillin-resistant and glycopeptide intermediate S. aureus strains in the data set. Differentiation based on the data in broader spectral regions was much less reliable. The results of this study indicate that with proper spectral region selection, FTIR spectroscopy and cluster analysis may provide a simple and accurate means of CNS species identification.     

Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Abstract Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium , on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.     

Use of Fourier transform infrared (FT-IR) spectroscopy as a tool for pollen identification

Airborne pollen are largely studied to obtain information about the atmospheric content of natural allergens. Aerobiological monitoring networks have been established to provide reliable data that facilitate the timely initiation of preventive actions aimed at minimizing allergic symptoms. Airborne pollen are usually identified and counted using an optical microscope, but as such procedures are extremely time-consuming, more expedient options are being explored. We have assessed the potential of Fourier transform infrared (FT-IR) spectroscopy as an alternative method for the rapid and reliable identification of allergenic pollen using six well-known allergenic pollen taxa and obtaining the respective FT-IR spectra. In doing this, a first IR spectral library has been created. The spectra of unknown pollen were compared to those of the reference library, and two pollen taxa of a mixed sample were identified.     

Phase behaviour and crystallinity of plant cuticular waxes studied by Fourier transform infrared spectroscopy

The phase behaviour of cuticular waxes from leaves of Hedera helix L. and Juglans regia L. was studied by Fourier transform infrared spectroscopy. For this purpose reconstituted waxes, isolated cuticular membranes, dewaxed polymer matrix membranes and whole leaves were studied in the horizontal attenuated total reflection and transmission modes. Melting curves of cuticular waxes were derived from temperature-dependent changes in the absorption maximum of the symmetric stretching mode of CH₂ groups (ν_s, at approx. 2856–2848 cm⁻¹). With increasing temperature absorption band doublets due to CH₂ scissoring (δ_sciss) and rocking (δ_rock) movements (at approx. 1473–1471 and 730–720 cm⁻¹, respectively) indicative of an orthorhombic arrangement of alkyl chains merged into a single peak. The area ratio of the peaks at approx. 720 and 730 cm⁻¹ was used as a measure for aliphatic crystallinity of plant cuticular waxes at a given temperature. The investigations of reconstituted cuticular waxes and those still embedded in isolated cuticles or in situ on the leaf produced comparable results. The findings are discussed in terms of the properties of the cuticular transport barrier. Received: 21 March 1997 / Accepted: 25 April 1997     

Differentiation of outer membrane proteins from Salmonellaenterica serotypes using Fourier transform infrared spectroscopy and chemometrics

AIMS: To differentiate between outer membrane proteins (OMPs) from six Salmonellaenterica serotypes using a Fourier transform infrared (FTIR) spectroscopy method and chemometrics. METHODS AND RESULTS: The OMPs from Salmonella serotypes (Typhimurium, Enteritidis, Thomasville, Hadar, Seftenberg and Brandenburg) were isolated using a sarcosyl extraction method. OMP profiles on SDS-PAGE exhibited two or three bands between 48 and 54 kDa. Spectra of 10 microl of OMP preparations (5 mg ml(-1)) dried on a gold reflective slide were collected using 128 scans at 4 cm(-1) resolution and units of log (1/R) and analyzed using canonical variate analysis (CVA) and linear discriminant analysis (LDA). The CVA of Salmonella OMP spectra in the 1800-1500 cm(-1) region separated the serotypes and LDA provided a 100% correct classification. CONCLUSIONS: The use of a FTIR method combined with chemometrics provided better differentiation of Salmonella OMPs than the OMP pattern analysis by SDS-PAGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that spectra of OMP extracts from Salmonella serotypes can be used for 100% correct classification of the serotypes studied.     

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.     

Dehydrating phospholipid vesicles measured in real-time using ATR Fourier transform infrared spectroscopy

According to the water replacement hypothesis, trehalose stabilizes dry membranes by preventing the decrease in spacing between adjacent phopspholipid headgroups during dehydration. Alternatively, the water-entrapment hypothesis postulates that in the dried state sugars trap residual water at the biomolecule sugar interface. In this study, Fourier transform infrared spectroscopy with an attenuated total reflection accessory was used to investigate the influence of trehalose on the dehydration kinetics and residual water content of egg phosphatidylcholine liposomes in real time under controlled relative humidity conditions. In the absence of trehalose, the lipids displayed a transition to a more ordered gel phase upon drying. The membrane conformational disorder in the dried state was found to decrease with decreasing relative humidity. Even at a relative humidity as high as 94% the conformational disorder of the lipid acyl chains decreased after evaporation of the bulk water. The presence of trehalose affects the rate of water removal from the system and the lipid phase behavior. The rate of water removal is decreased and the residual water content is higher, as compared to drying in the absence of trehalose. During drying, the level of hydrogen bonding to the head groups remains constant. In addition, the conformational disorder of the lipid acyl chains in the dried state more closely resembles that of the lipids in the fully hydrated state. We conclude that water entrapment rather than water replacement explains the effect of trehalose on lipid phase behavior of phosphatidylcholine lipid bilayers during the initial phase of drying.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Artificial neural network based identification of Campylobacter species by Fourier transform infrared spectroscopy

Two prototypes of artificial neural network (ANN), multilayer perceptron (MLP), and probabilistic neural network (PNN), were used to analyze infrared (IR) spectral data obtained from intact cells belonging to the species Campylobacter coli and Campylobacter jejuni. In order to establish a consistent identification and typing procedure, mid infrared spectra of these species were obtained by means of a Fourier transform infrared (FT-IR) spectroscope. FT-IR patterns belonging to 26 isolates subclassified into 4 genotypes were pre-processed (normalized, smoothed and derivatized) and grouped into training, verification and test sets. The two architectures tested (PNN, MLP) were developed and trained to identify or leave unassigned a number of IR patterns. Two window ranges (w(4), 1200 to 900 cm(-1); and w(5), 900 to 700 cm(-1)) in the mid IR spectrum were presented as input to the ANN models functioning as pattern recognition systems. No matter the ANN used all the training sets were correctly identified at subspecies level. For the test set, the four-layer MLP network was found to be specially suitable to recognize FT-IR data since it correctly identified 99.16% of unknowns using the w(4) range, and was fully successful in detecting atypical patterns from closely related Campylobacter strains and other bacterial species. The PNN network obtained lower percentages in assignation and rejection. Overall, ANNs constitute an excellent mathematical tool in microbial identification, since they are able to recognize with a high degree of confidence typical as well as atypical FT-IR fingerprints from Campylobacter spp.     

Probing conformational changes of proteins by quantitative second-derivative infrared spectroscopy

Probing protein conformational changes plays a crucial role in protein structure and function studies. However, the lack of efficient biophysical techniques makes it difficult to obtain the distinct behaviors of different secondary structure elements in a protein upon perturbation. This paper presents a discussion of the two major problems, the effect of sidelobes and different half-width at half-height (HWHH) values, encountered in quantitative second-derivative infrared (QSD-IR) spectroscopy and introduces the development of two criteria for checking the validity of the results obtained using the QSD-IR method. It was found that neither the sidelobes nor the HWHH significantly affected the quantitative result of protein conformational changes by using poly-l-lysine and hemoglobin as model proteins. A case study of bovine serum albumin (BSA) thermal aggregation suggested that the thermal transition of BSA was a process involving sequential events, and the two helical components were found to have a distinct response to heat perturbation. These results were confirmed by two-dimensional infrared correlation spectroscopy and by results in literature, suggesting that the QSD-IR method might be a potentially powerful tool to probe the distinct response of different secondary structures to perturbation.