Vibrational spectroscopy of seaweed galactans

Information from classical infrared spectroscopy studies has been of significance for characterizing seaweed galactans. The development of Fourier transform infrared spectroscopy and of Fourier transform laser Raman spectroscopy has produced great advances in the application of vibrational spectroscopy to the structural study of polysaccharides. Computational facilities in the spectrometers allow the arithmetic manipulations of the spectra. The second-derivative mode in the FT IR spectrocopy provided more information by increasing the number and resolution of the bands in the spectra as compared to the parent ones. A review of literature data on vibrational spectroscopy of sulfated polysaccharides and new results are presented. Agar-type polymers showed two diagnostic bands in the second-derivative mode in the region 800–700 cm^–1. Carrageenans exhibited a number of bands in the region 1600–1000 cm^–1. Fourier transform laser Raman spectroscopy in the solid state gave well-defined characteristic spectra of agar and carrageenans. Both techniques can be applied to small samples in the solid state and allow differentiation in a few minutes between agar and carrageenan-type seaweed galactans. The second-derivative mode of the FT IR spectra can be applied to distinguish agar-producing from carrageenan-producing seaweeds. The spectra on KBr pellets of dried, ground agarophyte and carrageenophyte seaweed samples showed the same bands as the corresponding polysaccharides.     

Hyperspectral and Thermal Imaging of Oilseed Rape (Brassica napus) Response to Fungal Species of the Genus Alternaria

In this paper, thermal (8-13 µm) and hyperspectral imaging in visible and near infrared (VNIR) and short wavelength infrared (SWIR) ranges were used to elaborate a method of early detection of biotic stresses caused by fungal species belonging to the genus Alternaria that were host (Alternaria alternata, Alternaria brassicae, and Alternaria brassicicola) and non-host (Alternaria dauci) pathogens to oilseed rape (Brassica napus L.). The measurements of disease severity for chosen dates after inoculation were compared to temperature distributions on infected leaves and to averaged reflectance characteristics. Statistical analysis revealed that leaf temperature distributions on particular days after inoculation and respective spectral characteristics, especially in the SWIR range (1000-2500 nm), significantly differed for the leaves inoculated with A. dauci from the other species of Alternaria as well as from leaves of non-treated plants. The significant differences in leaf temperature of the studied Alternaria species were observed in various stages of infection development. The classification experiments were performed on the hyperspectral data of the leaf surfaces to distinguish days after inoculation and Alternaria species. The second-derivative transformation of the spectral data together with back-propagation neural networks (BNNs) appeared to be the best combination for classification of days after inoculation (prediction accuracy 90.5%) and Alternaria species (prediction accuracy 80.5%).     

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.     

Peak locater for multicomponent spectra

A sliding pivot technique capable of locating component peak maxima of multicomponent spectra is presented. The locations of peak maxima obtained in this way are shown to be the same as those of the minima in the second derivative. A major advantage over the second-derivative test is simply that derivative spectra are not needed. The sliding pivot technique requires only the original spectrum to locate the component peak maxima and consequently reduces the noise enhancement factor. The deconvoluted Fourier transform infrared spectrum of purple membrane is analyzed and compared to a Gaussian analysis and a second-derivative analysis. The sliding pivot technique identifies a band missed by second-derivative analysis.     

Quantitative determination of tryptophanyl and tyrosyl residues of proteins by second-derivative fluorescence spectroscopy

Second-derivative spectroscopy has been applied to the study of the fluorescence of aromatic amino acids. The spectral features of the second derivative emission spectra of free aromatic amino acids and proteins are described, the emission of each aromatic fluorophore being characterized by a particular minimum-maximum pair. An easy, accurate, and rapid method is proposed for the quantitative determination of tyrosine and tryptophan, based on the addition of small amounts of a standard solution to the samples followed by the measurement of the increase in the distance between a selected minimum and an adjacent maximum, in the second-derivative spectrum. For tyrosine determination, excitation wavelength was 275 nm, and the selected minimum-maximum (m,M) pair was (300; 330 nm), while an excitation of 300 nm and a minimum-maximum pair (357; 377 nm) were employed for the tryptophan determination. This method enables the tryptophan content of proteins to be determined directly, without the need for correction for the presence of tyrosine. The tyrosine content of proteins can also be determined at neutral pH, in the presence of both tryptophan and phenylalanine. The proposed method has also been applied to trypsin activation of frog epidermis tyrosinase.     

Determination of compound aminopyrine phenacetin tablets by using artificial neural networks combined with principal components analysis

A method for simultaneous, nondestructive analysis of aminopyrine and phenacetin in compound aminopyrine phenacetin tablets with different concentrations has been developed by principal component artificial neural networks (PC-ANNs) on near-infrared (NIR) spectroscopy. In PC-ANN models, the spectral data were initially analyzed by principal component analysis. Then the scores of the principal components were chosen as input nodes for the input layer instead of the spectral data. The artificial neural network models using the spectral data as input nodes were also established and compared with the PC-ANN models. Four different preprocessing methods (first-derivative, second-derivative, standard normal variate (SNV), and multiplicative scatter correction) were applied to three sets of NIR spectra of compound aminopyrine phenacetin tablets. The PC-ANNs approach with SNV preprocessing spectra was found to provide the best results. The degree of approximation was performed as the selective criterion of the optimum network parameters.     

Electric field effects on the chlorophylls,pheophytins, and beta-carotenes in the reaction center of photosystem II

We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.     

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.     

Unfolding pathway of myoglobin: effect of denaturants on solvent accessibility to tyrosyl residues detected by second-derivative spectroscopy

The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.     

Inclusion complexes of N-sulfamoyloxazolidinones with beta-cyclodextrin

A study of inclusion complexes of six N-sulfamoyloxazolidinone derivatives with beta-cyclodextrin is described. The inclusion complexes were prepared in solution and in solid state with stoichiometry host-guest 1:1, and characterized. In solution, the complexation was carried out by spectrophotometric measurements at 25 degrees C. The stoichiometries and stability constants of complexes at various pHs have been determined using second-derivative spectrophotometry UV-vis. Hydrophobic properties of N-sulfamoyloxazolidinones are improved following their inclusion into beta-CD.     

Carbon tetrachloride, bromotrichloromethane and ethanol acute intoxication. New chemical evidence for lipid peroxidation in rat tissue microsomes.

Second-derivative spectroscopy was used to determine the conjugated-diene shift that measures the extent of the first step of lipid peroxidation after carbon tetrachloride (CCl4), bromotrichloromethane (BrCCl3) and ethanol intoxication. The conjugated-diene signal was recorded in the second-derivative spectra as a minimum peak at 233 nm. The use of this method enabled us to show that, under our experimental conditions, CCl4- and BrCCl3-dependent conjugated-diene formation in rat liver microsomes is not dose-dependent and increases linearly with time up to 3h. Proportionality was not obtained between the second-derivative-spectroscopy method, and the thiobarbituric acid and difference-spectra methods. In addition, whereas the thiobarbituric acid and difference-spectra methods gave positive results at zero time, second-derivative spectroscopy showed no evidence of formation of conjugated dienes under the same experimental conditions. Intoxication with ethanol was shown by the appearance of the conjugated-diene signal in liver microsomes 24 h after the administration of the toxin. Intoxication with either of the haloalkanes or ethanol did not give rise to any similar peak in lung and brain microsomes. The results obtained are discussed.     

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

Efficient Monte Carlo method for simulation of fluctuating conformations of native proteins

A powerful Monte Carlo method is described to simulate thermal conformational fluctuations in native proteins by using an empirical conformational energy function in which bond lengths and bond angles are kept fixed and only dihedral angles are independent variables. In this method, collective variables corresponding to eigenvectors of the second-derivative matrix of the energy function at its minimum point are scaled according to corresponding eigenvalues in such a way that the energy function in terms of the scaled collective variables is isotropic at the minimum point. Simulation is carried out with an isotropic step size in the space of these scaled collective variables. This simulation method is applied to a small protein, bovine pancreatic trypsin inhibitor (BPTI), and its model harmonic system defined by a quadratic energy function with the same second-derivative matrix as that of BPTI at its minimum point. Efficiency of the simulation method with an isotropic step size in the space of the scaled collective variables is found to be about 500–50 times greater than the conventional method with with an isotropic step in the space of the usual nonscaled variables. One step of this new method generates conformational changes that occur in the real-time range of 0.05 ps. In a record of 5 × 10⁵ step simulation, the BPTI molecule is observed to migrate beyond a single minimum-energy region.     

Long-distance cofactor interactions in terminal oxidases studied by second-derivative absorption spectroscopy

The electronic transitions of the two heme groups of cytochromec oxidase have been resolved by application of second-derivative and cryogenic absorption spectroscopy. Both methods reveal a splitting of the ferrocytochromea Soret transition into two features at 443 and 450 nm. The relative intensity of the 450 nm feature appears to depend on the ligation state of cytochromea ₃, the solution pH, and complex formation with cytochromec. The structural origin and mechanistic significance of this second Soret transition of cytochromea are discussed in terms of the electron transfer and proton translocation activities of the enzyme.Dedicated to the memory of James Carl Copeland.     

Arctic <Emphasis Type="Italic">Micromonas</Emphasis> uses protein pools and non-photochemical quenching to cope with temperature restrictions on Photosystem II protein turnover

Micromonas strains of small prasinophyte green algae are found throughout the world’s oceans, exploiting widely different niches. We grew arctic and temperate strains of Micromonas and compared their susceptibilities to photoinactivation of Photosystem II, their counteracting Photosystem II repair capacities, their Photosystem II content, and their induction and relaxation of non-photochemical quenching. In the arctic strain Micromonas NCMA 2099, the cellular content of active Photosystem II represents only about 50 % of total Photosystem II protein, as a slow rate constant for clearance of PsbA protein limits instantaneous repair. In contrast, the temperate strain NCMA 1646 shows a faster clearance of PsbA protein which allows it to maintain active Photosystem II content equivalent to total Photosystem II protein. Under growth at 2 °C, the arctic Micromonas maintains a constitutive induction of xanthophyll deepoxidation, shown by second-derivative whole-cell spectra, which supports strong induction of non-photochemical quenching under low to moderate light, even if xanthophyll cycling is blocked. This non-photochemical quenching, however, relaxes during subsequent darkness with kinetics nearly comparable to the temperate Micromonas NCMA 1646, thereby limiting the opportunity cost of sustained downregulation of PSII function after a decrease in light.     

Increased Drought Tolerance through the Suppression of ESKMO1 Gene and Overexpression of CBF-Related Genes in Arabidopsis

Improved drought tolerance is always a highly desired trait for agricultural plants. Significantly increased drought tolerance in Arabidopsis thaliana (Columbia-0) has been achieved in our work through the suppression of ESKMO1 (ESK1) gene expression with small-interfering RNA (siRNA) and overexpression of CBF genes with constitutive gene expression. ESK1 has been identified as a gene linked to normal development of the plant vascular system, which is assumed directly related to plant drought response. By using siRNA that specifically targets ESK1, the gene expression has been reduced and drought tolerance of the plant has been enhanced dramatically in the work. However, the plant response to external abscisic acid application has not been changed. ICE1, CBF1, and CBF3 are genes involved in a well-characterized plant stress response pathway, overexpression of them in the plant has demonstrated capable to increase drought tolerance. By overexpression of these genes combining together with suppression of ESK1 gene, the significant increase of plant drought tolerance has been achieved in comparison to single gene manipulation, although the effect is not in an additive way. Accompanying the increase of drought tolerance via suppression of ESK1 gene expression, the negative effect has been observed in seeds yield of transgenic plants in normal watering conditions comparing with wide type plant.     

API Determination by NIR Spectroscopy Across Pharmaceutical Production Process

The purpose of this research was to demonstrate the ability of reflectance near-infrared (NIR) spectroscopy for quantitative analysis of an active ingredient in different production steps of a solid formulation. The drug is quantified at two different steps of a pharmaceutical process: after granulation and after tablet coating. Calibration samples were prepared by mixing pure drug, excipients, and batch samples (75–120 mg/g active ingredient) using a simple methodology that can be easily carried out in a laboratory. Partial least squares calibration models were calculated in second-derivative mode using the wavelength range 1,134–1,798 nm. The error of prediction for granulated samples was 1.01% and 1.63% for tablets. The results prove that NIR spectroscopy is a good alternative to other, more time-consuming means of analysis for pharmaceutical process monitoring.     

Infrared study of human serum very-low-density and low-density lipoproteins. Implication of esterified lipid C=O stretching bands for characterizing lipoproteins

Fourier-transform infrared (FT-IR) spectroscopy was applied to examine human serum very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in aqueous solution and in solid film for characterizing lipid components. On the basis of the FT-IR second-derivative spectra for standard samples of triglycerides, cholesterol esters and phospholipids, it was found that the band at 1746 cm(-1) for VLDL and the band at 1738 cm(-1) for LDL were mainly due to the unsaturated triglycerides and unsaturated cholesterol esters, respectively. The implications of ester C=O stretching bands are discussed.