The phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as seen by Fourier transform infrared difference spectroscopy

The potential of Fourier transform infrared difference spectroscopy for biochemical applications is demonstrated by the gel to liquid crystal phase transition of the title compound. While the changes occurring in the vibrational pattern of the hydrophobic palmitoyl chains are easily monitored, this technique also discriminates between no change in the choline moiety and a small yet significant change in the carbonyl moiety, both located in the hydrophylic head group.     

Lipid reorganization in biological membranes. A study by fourier transform infrared difference spectroscopy

The first application of infrared difference spectroscopy to the study of a natural biological membrane is described. Perdeuterated palmitic acid was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii and the temperature-induced structural rearrangement of the endogenous lipids monitored via their C²H vibrational modes. Changes in infrared parameters were studied between 0 and 50°C and contrasted with those occurring in the model membrane system of $\text{1,2-}\text{diperdeuteropalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. The phase transition of the biomembrane occurs over a 20°C range with the temperature of the maximum rate of change of absorbance coinciding with that of the sharp phase transition of the model membrane.     

Discrimination of geographical origin and adulteration of radix astragali using fourier transform infrared spectroscopy and chemometric methods

Introduction – Radix Astragali, one of most widely used and important traditional Chinese medicines, is cultivated in different geographical regions. Because of varying growing conditions, the qualities of Radix Astragali vary, which can give rise to differences in clinical therapy. Detecting adulteration is a routine requirement in pharmaceutical practice. Objective – To develop a simple and accurate approach to discriminate the geographical origin and potential adulteration of Radix Astragali, derived from the root of Astragalus membranaceus (Fischer) Bunge var. mongholicus (Bunge) Hsiao, using Fourier transform infrared (FT‐IR) spectroscopy and chemometric methods. Methodology – To obtain characteristic IR spectra for accurate discrimination, a one‐solvent extraction method was utilised following a novel evaluation method for selecting appropriate solvents. Samples of Radix Astragali from different geographical origins were discriminated using FT‐IR spectroscopy and discriminant partial least squares (DPLS) methods. FT‐IR spectroscopy combined with Mahalanobis distance was employed to detect adulteration of Radix Astragali. Results – In comparison with other solvents, butanone was more effective at extracting samples. Radix Astragali samples were accurately assigned to their corresponding geographical origins by using FT‐IR spectroscopy and DPLS method. Most adulterated samples were detected accurately by application of FT‐IR spectroscopy combined with Mahalanobis distance. Conclusion – FT‐IR spectroscopy combined with chemometric method was developed and demonstrated to be a useful tool to discriminate geographical origin and adulteration of Radix Astragali. Copyright © 2010 John Wiley & Sons, Ltd.     

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 monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

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.     

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: a Fourier transform infrared spectroscopy study

Arteriovenous malformations (AVMs), masses of abnormal blood vessels which grow in the brain, produce high flow shunts that steal blood from surrounding brain tissue, which is chronically hypoperfused. Hypoperfusion is a condition of inadequate tissue perfusion and oxygenation, resulting in abnormal tissue metabolism. Fourier transform infrared (FTIR) spectroscopy is used in this study to investigate the effect of hypoperfusion on homogenized rat brain samples at the molecular level. The results suggest that the lipid content increases, the protein content decreases, the lipid-to-protein ratio increases, and the state of order of the lipids increases in the hypoperfused brain samples. FTIR results also revealed that, owing to hypoperfusion, not only the protein synthesis but also the protein secondary structure profile is altered in favor of -sheets and random coils. These findings clearly demonstrate that, FTIR spectroscopy can be used to extract valuable information at the molecular level so as to have a better understanding of the effect of hypoperfusion on rat brain.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

Variability of protein and lipid composition of human subtantia nigra in aging: Fourier transform infrared microspectroscopy study

There is growing evidence that a variety of biochemical processes that underlie the most frequent neurodegenerative diseases may have much in common with those connected with natural aging. It was shown that they involve, among others, lipid peroxidation and/or generation of insoluble in water protein deposits (i.e. alpha-synuclein and/or beta amyloid). Therefore, it is likely that the analysis of changes in both lipid and protein composition may be interesting in the light of any potential pathologies occurring within the dopaminergic system during physiological aging. Thereby, this paper presents a methodology for the analysis of age-related changes in a lipid and protein composition within human subtantia nigra tissue by means of Fourier transform infrared microspectroscopy (FTIRM). Particularly, the changes in the lipid saturation, unsaturation as well as in the protein secondary structure were examined. The studies were carried out on samples from 35 individuals who died without any signs of neurologic dysfunctions. Our results show that the level of lipid saturation increases inside the subtantia nigra tissue with age, though the total content of lipid decreases with age of individuals. Moreover, the statistically significant decrease in the protein content within neuron bodies was observed. Interestingly, it is presented that the content of the anti-parallel beta sheets for neuron bodies decreases from seventh to eighth decades of life and subsequently markedly increases from eighth to ninth decades of life, whilst, as regards extraneuronal spaces, the opposite trends are reported i.e. increase from the seventh to eighth decades, and subsequent decrease in the ninth decade of life. These observations, though preliminary, shed the light on a putative contribution of various pathological lipid- and protein-related processes underlying senescence, suggesting a “biochemical link” between the aetiology of the most common neurodegenerative diseases and physiological aging.     

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.     

畜禽粪便堆肥的理化腐熟指标及其红外光谱

选择新鲜的牛粪、猪粪和鸡粪为堆肥原料,通过室内模拟直接堆肥试验,研究堆肥过程理化参数变化、种子发芽指数以及腐熟物红外光谱特征.结果表明: 堆肥过程温度变化明显,牛粪出现一次堆肥高温阶段,鸡粪和猪粪各两次,堆肥温度超过50 ℃以上时间均大于10 d,温度可以最直观地表征堆肥的腐熟程度.堆肥过程中pH值变化幅度较大,其中猪粪的pH值从6.63上升至7.74,鸡粪从7.73上升至8.66,牛粪则先从7.86上升至8.36再下降至7.52;有机碳含量逐渐降低,牛粪、猪粪和鸡粪分别下降23.3%、28.2%和31.7%;堆肥过程中,牛粪、猪粪和鸡粪的铵态氮含量分别下降87.8%、73.6%和79.7%,硝态氮含量分别增加至堆肥前的56.81、6.49和4.85倍,铵态氮/硝态氮均下降至2以下.温度、pH值、有机碳含量和铵态氮/硝态氮能较好地反映3种粪便高温堆肥的腐熟程度,且与种子发芽率相关性较高(P≤0.05).红外光谱分析表明,在堆肥过程中,牛粪和猪粪的脂肪族、多糖类物质减少,芳香结构化增强,腐殖化程度增加;鸡粪则相反.在建立理化指标体系的同时,应兼顾物质结构的稳定性.     

Melatonin strongly interacts with zwitterionic model membranes—evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry

Interactions of melatonin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and melatonin concentration (1-30 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The investigation of the C-H, CO, and PO₂⁻ antisymmetric double stretching modes in FTIR spectra and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, and increasing the dynamics of the system both in the gel and liquid crystalline phases. It also causes significant decrease in the wavenumber for the CO stretching and PO₂⁻ antisymmetric double bond stretching bands, which indicates strong hydrogen bonding The results imply that melatonin locates in the interfacial region of the membrane. Furthermore, in the DSC curve, more than one signal is observed at high melatonin concentrations (24 and 30 mol%), which indicates melatonin-induced phase separation in DPPC membranes.     

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm⁻¹ for scl-PHA and at 2933 and 2976 cm⁻¹ for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm⁻¹ for HV-copolymer. The copolymers were analyzed by GC and ¹H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.     

Fourier‐transform infrared spectroscopy study of dehydrated lipases from Candida antarctica B and Pseudomonas cepacia

Fourier‐transform infrared (FT‐IR) spectroscopy was employed to investigate potential lyophilization‐induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the α‐helix and increased the β‐sheet content. However, FT‐IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the β‐sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT‐IR for analysis of the structure of solid proteins, at least as far as the β‐sheet content is concerned, because it is possible that the method overestimates the β‐sheets by measuring other hydrogen‐bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 545–551, 1999.     

Molecular interactions of the redox-active accessory chlorophyll on the electron-donor side of photosystem II as studied by Fourier transform infrared spectroscopy

A Fourier transform infrared (FTIR) difference spectrum upon photooxidation of the accessory chlorophyll (Chl_z) of photosystem II (PS II) was obtained at 210 K with Mn-depleted PS II membranes in the presence of fericyanide and silicomolybdate. The observed Chl_z⁺/Chl_z spectrum showed two differential bands at 1747/1736 and 1714/1684 cm⁻. The former was assigned to the free carbomethoxy C = 0 and the latter to the keto C = 0 that is hydrogen-bonded or in a highly polar environment. Also, the negative 1614 cm⁻ band assignable to the macrocycle mode indicated 5-coordination of the central Mg. The negative 1660 cm⁻¹ band, possibly due to the strongly hydrogen-bonded keto C = 0, may suggest oxidation of one more Chl_z, although an alternative assignment, the amide I mode of proteins perturbed by Chl_z oxidation, is also possible.