The effect of the fat content on the thermal effusivity of foods: an inverse photopyroelectric study
Szafner, G. ; Bicanic, D.D. ; Doka, O. - \ 2011
International journal of Food Properties 14 (2011)3. - ISSN 1094-2912 - p. 666 - 674.
rheological characterization - light mayonnaises - milk - spectroscopy - acids - dsc
Photopyroelectric (PPE) methods belong to the class of photothermal techniques and provide the means for determining some thermal properties of foods in a relatively fast and simple way. In particular, the inverse variant of the photopyroelectric method, abbreviated IPPE, was used here to determine thermal effusivity (also called heat penetration coefficient) of the sour cream and mayonnaise as a function of their fat content. In the sour cream the latter varied from 12 to 31 g/100 g as compared to 27 to 80 g/100 g range in mayonnaise; for both samples the effusivity decreased linearly with the increasing fat content. Each additional gram of fat in 100 g sour cream or mayonnaise resulted in 11.13 and 12.11 Ws1/2m-2K-1 drop in effusivity. Good agreement between the experimentally obtained data and the calculated effusivity was observed if both, the composition and the thermal properties of individual constituents of sour cream were known.
Structural features and properties of soluble products derived from Eucalyptus globulus hemicelluloses
Gullon, P. ; González-Muñoz, M.J. ; Gool, M.P. van; Schols, H.A. ; Hirsch, J. ; Ebringerová, A. ; Parajo, J.C. - \ 2011
Food Chemistry 127 (2011)4. - ISSN 0308-8146 - p. 1798 - 1807.
xylo-oligosaccharides - in-vitro - autohydrolysis liquors - intestinal microbiota - wood - xylooligosaccharides - fermentability - fermentation - spectroscopy - manufacture
Eucalyptus globulus wood samples were subjected to double hydrothermal processing to remove extractives in the first stage, and to cause the selective solubilisation of 4-O-methylglucuronoxylan in the second stage. The hemicellulose-derived products present in the liquors from the second hydrothermal stage (substituted xylooligosaccharides, denoted XOS) were refined by treatments with membranes and ion exchange. The purified XOS product was assayed for composition and characterised by HPLC-RI, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR techniques. The results suggested the presence of neutral and acidic XOS with a degree of acetylation of about 0.6. The fermentability of the refined XOS product by faecal inocula was assessed by measuring both substrate consumption and formation of short-chain fatty acids.
A general approach for detecting folding intermediates from staedy-state and time-resolved fluorescence of single-tryptophan-containing proteins
Laptenok, S. ; Visser, N.V. ; Ruchira, A. ; Westphal, A.H. ; Hoek, A. van; Mierlo, C.P.M. van; Stokkum, I.H.M. van; Amerongen, H. van; Visser, A.J.W.G. - \ 2011
Biochemistry 50 (2011)17. - ISSN 0006-2960 - p. 3441 - 3450.
azotobacter-vinelandii - apoflavodoxin - pathway - spectroscopy - peptide
During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is 4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin
The patterns of population differentiation in a Brassica rapa core collection
Pino del Carpio, D. ; Basnet, R.K. ; Vos, R.C.H. de; Maliepaard, C.A. ; Visser, R.G.F. ; Bonnema, A.B. - \ 2011
Theoretical and Applied Genetics 122 (2011)6. - ISSN 0040-5752 - p. 1105 - 1118.
genetic diversity - morphological traits - l. - association - aflp - microsatellites - spectroscopy - metabolomics - frequencies - landraces
With the recent advances in high throughput profiling techniques the amount of genetic and phenotypic data available has increased dramatically. Although many genetic diversity studies combine morphological and genetic data, metabolite profiling has yet to be integrated into these studies. For our study we selected 168 accessions representing the different morphotypes and geographic origins of Brassica rapa. Metabolite profiling was performed on all plants of this collection in the youngest expanded leaves, 5 weeks after transplanting and the same material was used for molecular marker profiling. During the same season a year later, 26 morphological characteristics were measured on plants that had been vernalized in the seedling stage. The number of groups and composition following a hierarchical clustering with molecular markers was highly correlated to the groups based on morphological traits (r = 0.420) and metabolic profiles (r = 0.476). To reveal the admixture levels in B. rapa, comparison with the results of the programme STRUCTURE was needed to obtain information on population substructure. To analyze 5546 metabolite (LC–MS) signals the groups identified with STRUCTURE were used for random forests classification. When comparing the random forests and STRUCTURE membership probabilities 86% of the accessions were allocated into the same subgroup. Our findings indicate that if extensive phenotypic data (metabolites) are available, classification based on this type of data is very comparable to genetic classification. These multivariate types of data and methodological approaches are valuable for the selection of accessions to study the genetics of selected traits and for genetic improvement programs, and additionally provide information on the evolution of the different morphotypes in B. rapa
Annual balances of CH4 and N2O from a managed fen meadow using eddy covariance flux measurements
Kroon, P.S. ; Schrier-Uijl, A.P. ; Hensen, A. ; Veenendaal, E.M. ; Jonker, H.J.J. - \ 2010
European Journal of Soil Science 61 (2010)5. - ISSN 1351-0754 - p. 773 - 784.
nitrous-oxide emissions - methane emission - peat soils - micrometeorological techniques - grassland systems - carbon - exchange - climate - spectroscopy - netherlands
Annual terrestrial balances of methane (CH4) and nitrous oxide (N2O) are presented for a managed fen meadow in the Netherlands for 2006, 2007 and 2008, using eddy covariance (EC) flux measurements. Annual emissions derived from different methods are compared. The most accurate annual CH4 flux is achieved by gap filling EC fluxes with an empirical multivariate regression model, with soil temperature and mean wind velocity as driving variables. This model explains about 60% of the variability in observed daily CH4 fluxes. Annual N2O emissions can be separated into background emissions and event emissions due to fertilization. The background emission is estimated using a multivariate regression model also based on EC flux data, with soil temperature and mean wind velocity as driving variables. The event emissions are estimated using emission factors. The minimum direct emission factor is derived for six fertilization events by subtracting the background emission, and the IPCC default emission factor of 1% is used for the other events. In addition, the maximum direct emission factors are determined for the six events without subtracting the background emission. The average direct emission factor ranges from 1.2 to 2.8%, which is larger than the IPCC default value. Finally, the total terrestrial greenhouse gas balance is estimated at 16 Mg ha-1 year-1 in CO2-equivalents with contributions of 30, 25 and 45% by CO2, CH4 and N2O, respectively.
ATP Changes the Fluorescence Lifetime of Cyan Fluorescent protein via an Interaction with His148
Borst, J.W. ; Willemse, M. ; Slijkhuis, R. ; Krogt, G. ; Laptenok, S. ; Jalink, K. ; Wieringa, B. ; Fransen, J.A.M. - \ 2010
PLoS ONE 5 (2010)11. - ISSN 1932-6203 - 7 p.
energy-transfer - variant - fret - cell - spectroscopy - chromophore - binding - decays
Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.
Carotenoid fluorescence in Dunaliella salina
Kleinegris, D.M.M. ; Es, M.A. van; Janssen, M.G.J. ; Brandenburg, W.A. ; Wijffels, R.H. - \ 2010
Journal of Applied Phycology 22 (2010)5. - ISSN 0921-8971 - p. 645 - 649.
beta-carotene - spectroscopy - pigments
Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content.
Electrical double-layer capacitance in room temperature ionic liquids: Ion size and specific adsorption effects
Lauw, Y. ; Horne, M.D. ; Rodopoulos, T. ; Nelson, A. ; Leermakers, F.A.M. - \ 2010
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 114 (2010). - ISSN 1520-6106 - p. 11149 - 11154.
interacting chain molecules - enhanced raman-scattering - sum-frequency generation - partial charge-transfer - differential capacitance - statistical-theory - surface-structure - electrode - interface - spectroscopy
The electrical double-layer structure and capacitance in room temperature ionic liquids at electrified interfaces were systematically studied with use of the self-consistent mean-field theory. The capacitance curve departs from symmetry with respect to the point of zero charge when unequal ion-size is implemented or when specific adsorption of ions is introduced. For the case of unequal ion-size, the shape of the capacitance curve is strongly determined by the size of the counterion and only weakly influenced by the co-ion size. When present, specifically adsorbed ions would change the capacitance within a limited range of applied potential from the point of zero charge, which itself varies with the strength of specific adsorption.
Monitoring photosynthesis in individual cells of Synechocytis sp. PCC 6803 on a picosecond timescale
Krumova, S.K.B. ; Laptenok, S. ; Borst, J.W. ; Ughy, B. ; Gombos, Z. ; Aijani, G. ; Amerongen, H. van - \ 2010
Biophysical Journal 99 (2010)6. - ISSN 0006-3495 - p. 2006 - 2015.
fluorescence emission-spectra - excitation-energy transfer - intact photosystem-ii - thylakoid membrane - synechococcus 6301 - phycobilisome - kinetics - time - spectroscopy - chlorophyll
Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: 20 ps (PSI and PSII), 80 ps, 440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future
High molecular weight glucan of the culinary medicinal mushroom Agaricus bisporus is an a-glucan that forms complexes with low molecular weight galactan
Smiderle, F. ; Sassaki, G.L. ; Arkel, J. van; Lacomini, M. ; Wichers, H.J. ; Griensven, L.J.L.D. van - \ 2010
Molecules 15 (2010)8. - ISSN 1420-3049 - p. 5818 - 5830.
beta-glucans - flammulina-velutipes - edible mushroom - in-vitro - polysaccharide - pleurotus - extracts - glycogen - purification - spectroscopy
An a-glucan was isolated from the culinary medicinal mushroom A. bisporus by hot water extraction, ethanol precipitation and DEAE-cellulose chromatography. The resulting material showed a single HMW peak excluded from a Sephadex G50 column that could completely be degraded by a-amylase treatment. After heating in 1% SDS a small additional peak of low MW eluted from the G50 column. The monosaccharide composition of the main peak was evaluated by HPLC, and was found to consist of a majority of glucose (97.6%), and a minor proportion of galactose (2.4%). Methylation analysis and degradation by a-amylase indicated the presence of an a-glucan with a main chain consisting of (1®4)-linked units, substituted at O-6 by a-D-glucopyranose single-units in the relation 1:8. Mono- (13C-, 1H-NMR) and bidimensional [1H (obs.),13C-HSQC] spectroscopy analysis confirmed the a-configuration of the Glcp residues by low frequency resonances of C-1 at d 100.6, 100.2, and 98.8 ppm and H-1 high field ones at d 5.06, 5.11, and 4.74 ppm. The DEPT-13C-NMR allowed assigning the non-substituted and O-substituted –CH2 signals at d 60.3/60.8 and 66.2 ppm, respectively. Other assignments were attributed to C-2, C-3, C-4, C-5 and C-6 of the non-reducing ends at d 71.8; 72.8; 70.0; 71.3 and 60.3/60.8 ppm, respectively. The minor proportion of galactose that was demonstrated was probably derived from a complex between the a-glucan and a low molecular weight galactan
Nanowires Formed by the Co-Assembly of a Negatively Charged Low-Molecular Weight Gelator and a Zwitterionic Polythiophene
Li, F. ; Ganesan, P. ; Jong, M.R. de; Aslund, A. ; Konradsson, P. ; Marcelis, A.T.M. ; Sudhölter, E.J.R. ; Cohen Stuart, M.A. ; Leermakers, F.A.M. - \ 2010
ChemPhysChem 11 (2010). - ISSN 1439-4235 - p. 1956 - 1960.
conjugated polyelectrolytes - spectroscopy - derivatives - organogels - alignment
Conjugated organic nanowires have been prepared by co-assembling a carboxylate containing low-molecular weight gelator (LMWG) and an amino acid substituted polythiophene derivative (PTT). Upon introducing the zwitterionic polyelectrolyte PTT to a basic molecular solution of the organogelator, the negative charges on the LMWG are compensated by the positive charges of the PTT. As a result, nanowires form through co-assembly. These nanowires are visualized by both transmission electron microscopy (TEM) and atomic force microscopy (AFM). Depending on the concentration and ratio of the components these nanowires can be micrometers long. These measurements further suggest that the aggregates adopt a helical conformation. The morphology of these nanowires are studied with fluorescent confocal laser scanning microscopy (CLSM). The interactions between LMWG and PTT are characterized by steady-state and time-resolved fluorescence spectroscopy studies. The steady-state spectra indicate that the backbone of the PTT adopts a more planar and more aggregated conformation when interacting with LMWG. The time-resolved fluorescence decay studies confirm this interpretation.
Estimating rapidly and precisely the concentration of beta carotene in mango homogenates by measuring the amplitude of optothermal signals, values of chromaticity indices and the intensities of Raman peaks
Bicanic, D.D. ; Dimitrovski, D. ; Luterotti, S. ; Tiwisk, C. van; Buijnsters, J.G. ; Doka, O. - \ 2010
Food Chemistry 121 (2010)3. - ISSN 0308-8146 - p. 832 - 838.
liquid-chromatography - lycopene - window - quantification - spectroscopy - vegetables - separation - fruits - hplc
Rapid, quantitative information about the micronutrients (including beta carotene) in mango fruit is often desired. High performance liquid chromatography (HPLC) and spectrophotometry (SP), the two widely used methods in practice to quantify carotenoids, both require a time consuming and expensive extraction of a pigment prior to the analysis itself. This paper compares the performances of the three candidate methods for the assessment of beta carotene in twenty one different mango homogenates to that of the HPLC as an established standard technique. The extraction is imperative in neither of the methods: the laser based optothermal window (OW), the resonance Raman spectroscopy and the tristimulus colorimetry. For the quantitative analysis however the availability of the calibration curve is a necessity. All candidate methods and in particular OW technique (compact instrument, low cost and the ease of operation) hold promise for a rapid screening/quantitative assessment of beta carotene in mango fruit
Authentication of feeding fats: Classification of animal fats, fish oils and recycled cooking oils
Ruth, S.M. van; Rozijn, M. ; Koot, A.H. ; Perez-Garcia, R. ; Kamp, H.J. van der; Codony, R. - \ 2010
Animal Feed Science and Technology 155 (2010)1. - ISSN 0377-8401 - p. 65 - 73.
reaction-mass-spectrometry - partial least-squares - trace gas-analysis - electronic nose - vegetable-oils - discrimination - spectroscopy - acids
Classification of fats and oils involves the recognition of one/several markers typical of the product. The ideal marker(s) should be specific to the fat or oil. Not many chemical markers fulfill these criteria. Authenticity assessment is a difficult task, which in most cases requires the measurement of several markers and must take into account natural and technology-induced variation. The present study focuses on the identity prediction of three by-products of the fat industry (animal fats, fish oils, recycled cooking oils), which may be used for animal feeding. Their identities were predicted by their triacylglycerol fingerprints, their fatty acid fingerprints and their profiles of volatile organic compounds. Partial least square discriminant analysis allowed samples to be assigned successfully into their identity classes. Most successful were triacylglycerol and fatty acid fingerprints (both 96% correct classification). Proton transfer reaction mass spectra of the volatile compounds predicted the identity of the fats in 92% of the samples correctly.
Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins
Vos, W.L. ; Schor, M. ; Baumgaertner, A. ; Tieleman, D.P. ; Hemminga, M.A. - \ 2010
European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 229 - 239.
major coat protein - transmembrane alpha-helix - energy-transfer - fret - orientation - conformation - spectroscopy - model - association - bilayers
Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, a-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein
Remote sensing of sun-induced fluorescence to improve modeling of diurnal courses of gross primary production (GPP)
Damm, A. ; Elbers, J.A. ; Erler, A. ; Giolis, B. ; Hamdi, K. ; Hutjes, R.W.A. ; Kosvancova, M. ; Meroni, M. ; Migliettas, F. ; Moersch, A. ; Moreno, J. ; Schickling, A. ; Sonnenschein, R. ; Udelhoven, T. ; Linden, S. van der; Hostert, P. ; Rascher, U. - \ 2010
Global Change Biology 16 (2010)1. - ISSN 1354-1013 - p. 171 - 186.
koolstofcyclus - primaire productie - remote sensing - fluorescentie - spectroscopie - planten - fotosynthese - modelleren - carbon cycle - primary production - remote sensing - fluorescence - spectroscopy - plants - photosynthesis - modeling - light-use efficiency - induced chlorophyll fluorescence - photochemical reflectance index - net primary production - eddy covariance - photosynthetic efficiency - leaf senescence - photosystem-ii - carbon-dioxide - boreal forest
Terrestrial gross primary production (GPP) is an important parameter to explore and quantify carbon fixation by plant ecosystems at various scales. Remote sensing (RS) offers a unique possibility to investigate GPP in a spatially explicit fashion; however, budgeting of terrestrial carbon cycles based on this approach still remains uncertain. To improve calculations, spatio-temporal variability of GPP must be investigated in more detail on local and regional scales. The overarching goal of this study is to enhance our knowledge on how environmentally induced changes of photosynthetic light-use efficiency (LUE) are linked with optical RS parameters. Diurnal courses of sun-induced fluorescence yield (FSyield) and the photochemical reflectance index of corn were derived from high-resolution spectrometric measurements and their potential as proxies for LUE was investigated. GPP was modeled using Monteith's LUE-concept and optical-based GPP and LUE values were compared with synoptically acquired eddy covariance data. It is shown that the diurnal response of complex physiological regulation of photosynthesis can be tracked reliably with the sun-induced fluorescence. Considering structural and physiological effects, this research shows for the first time that including sun-induced fluorescence into modeling approaches improves their results in predicting diurnal courses of GPP. Our results support the hypothesis that air- or spaceborne quantification of sun-induced fluorescence yield may become a powerful tool to better understand spatio-temporal variations of fluorescence yield, photosynthetic efficiency and plant stress on a global scale
Global analysis of multiple gas chromatography-mass spectrometry (GC/MS) data sets: A method for resolution of co-eluting components with comparison to MCR-ALS
Stokkum, I.H.M. van; Mullen, K.M. ; Mihaleva, V.V. - \ 2009
Chemometrics and Intelligent Laboratory Systems 95 (2009)2. - ISSN 0169-7439 - p. 150 - 163.
multivariate curve resolution - nonlinear least-squares - compound identification - liquid-chromatography - feasible solutions - spectral library - band boundaries - maximum - samples - spectroscopy
Global analysis has been applied to resolve components in multiple gas chromatography-mass spectrometry (GC/MS) data sets. Global analysis methodology is based upon a parametrized model of the observed data, including random (and possibly also systematic) errors. Each elution profile is described as a function of a small number of parameters. We successfully based the description of elution profiles on an exponentially modified Gaussian. The mass spectra were described non-parametrically. Model usefulness is judged by the quality of the fit and whether the estimated parameters that describe the elution profiles and mass spectra of components are physically interpretable. Advantages of the method are most evident with multiple data sets and overlapping elution profiles. Differences between data sets are described by alignment parameters and by relative amplitude parameters. The estimated mass spectrum is identical between experiments. Global analysis and multivariate curve resolution alternating least squares (MCR-ALS) are the only methods currently developed for component resolution for the case of completely co-eluting compounds in mass spectrometry data. In the present contribution global analysis is shown to have better performance than MCR-ALS in terms of the estimated mass spectra for a variety of simulated GC mass spectrometry datasets representing components that are completely co-eluting.
Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness
Stopar, D. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Biochimica et Biophysica Acta. Biomembranes 1788 (2009)10. - ISSN 0005-2736 - p. 2217 - 2221.
membrane-proteins - tryptophan residues - amino-acids - peptides - topology - helix - spectroscopy - orientation
Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less ( 70 cm-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain
Identification of Uranyl Surface Complexes an Ferrihydrite: Advanced EXAFS Data Analysis and CD-MUSIC Modeling
Rossberg, A. ; Ulrich, K.U. ; Weiss, S. ; Tsushima, S. ; Hiemstra, T. ; Scheinost, A.C. - \ 2009
Environmental Science and Technology 43 (2009)5. - ISSN 0013-936X - p. 1400 - 1406.
transformation factor-analysis - uranium(vi) sorption - adsorption - acid - hematite - spectroscopy - goethite - u(vi)
Previous spectroscopic research suggested that uranium(VI) adsorption to iron oxides is dominated by ternary uranyl-carbonato surface complexes across an unexpectedly wide pH range. Formation of such complexes would have a significant impact on the sorption behavior and mobility of uranium in aqueous environments. We therefore reinvestigated the identity and structural coordination of uranyl sorption complexes using a combination of U LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and iterative transformation factor analysis, which enhances the resolution in comparison to conventional EXAFS analysis. A range of conditions (pH, CO2 partial pressure, ionic strength) made it possible to quantify the variations in surface speciation. In the resulting set of spectral data (N = 11) the variance is explained by only two components, which represent two structurally different types of surface complexes: (1) a binary uranyl surface complex with a bidentate coordination to edges of Fe(O,OH)6 octahedra and (2) a uranyl triscarbonato surface complex where one carbonate ion bridges uranyl to the surface. This ternary type B complex differs from a type A complex where uranyl is directly attached to surface atoms and carbonate is bridged by uranyl to the surface. Both surface complexes agree qualitatively and quantitatively with predictions by a charge distribution (CD) model. According to this model the edge-sharing uranyl complex has equatorial ligands (-OH2, -OH, or one -CO3 group) that point away from the surface. The monodentate uranyl triscarbonato surface complex (type B) is relevant only at high pH and elevated pCO2. At these conditions, however, it is responsible for significant uranyl sorption, whereas standard models would predict only weak sorption. This paper presents the first spectroscopic evidence of this ternary surface complex, which has significant implications for immobilization of uranyl in carbonate-rich aqueous environments
Modeling membrane protein structure through site-directed ESR spectroscopy
Kavalenka, A.A. - \ 2009
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga; J. Strancar. - [S.l. : S.n. - ISBN 9789085854241 - 119
oppervlakte-eiwitten - moleculaire structuur - spectroscopie - paramagnetische elektronenresonantiespectroscopie - surface proteins - molecular conformation - spectroscopy - electron paramagnetic resonance spectroscopy
Site-directed spin labeling (SDSL) electron spin resonance (ESR) spectroscopy is a
relatively new biophysical tool for obtaining structural information about proteins. This
thesis presents a novel approach, based on powerful spectral analysis techniques (multicomponent
spectral simulations and evolutionary optimizations of ESR spectra) and
modeling of the protein structure by calculating the restrictions of the conformational space
of the attached spin label.
First, the feasibility of the ESR spectral analysis was enhanced by speeding-up the
spectrum optimization and by automation of the analysis routines to enable the handling of
large sets of spectroscopic data (e.g., for the joint analysis of SDSL-ESR spectra from
multiple sites of a spin-labeled protein). According to the testing examples a speed-up
factor of 5-7 was achieved.
Secondly, SDSL-ESR was used to study the topology of the long N-terminal domain
of the photosynthetic light-harvesting complex CP29. Wild-type protein containing a single
cysteine at position 108 and nine single cysteine mutants were produced, allowing to label
different parts of the domain with a nitroxide spin label. In all cases the apoproteins were
either solubilized in detergent, or they were reconstituted with their native pigments in
vitro. The spin label ESR spectra were analyzed in terms of a multi-component spectral
simulation approach. These results permit to trace the structural organization of the long Nterminal
domain of CP29 leading to a structural model for its N-terminal domain.
Thirdly, we proposed a novel way to translate the local structural constraints gained
by SDSL-ESR data into a low-resolution structure of a protein by simulating the
restrictions of the local conformational spaces of the spin label attached at different protein
sites along the primary structure of the membrane-embedded protein. The proposed
structural model takes into account the restricting effect of the protein backbone, amino
acid side chains and lipid environment. We tested the sensitivity of this approach for
artificial oligopeptides and then for membrane-embedded M13 major coat protein
decorated with a limited number of strategically placed spin labels by employing highthroughput
site-directed mutagenesis. We found a reasonably good agreement of the
simulated and the experimental data taking a protein conformation close to an α-helix.
Finally, by using an optimization algorithm we optimized the parameters of the
protein-lipid model by improving the fit of the simulation data to the experimental
conformational space data. The outcome of the optimization was a family of best-fit
structures of membrane-embedded M13 protein, which not only agree with the available
SDSL-ESR data, but also was consistent with a recent model based on site-directed
Therefore, the present method provides a challenging starting point for the
development of a powerful methodology for the protein structure characterization, an
alternative approach to conventional techniques.
The influence of vegetation cover on the spectroscopic estimation of soil properties
Bartholomeus, H. - \ 2009
Wageningen University. Promotor(en): Michael Schaepman, co-promotor(en): Lammert Kooistra. - [S.l. : S.n. - ISBN 9789085854487 - 144
bodemeigenschappen - vegetatie - spectroscopie - schatting - koolstof - landbouwgronden - ijzer - bodemchemie - geostatistiek - soil properties - vegetation - spectroscopy - estimation - carbon - agricultural soils - iron - soil chemistry - geostatistics
Voor het bepalen van de kwaliteit van de bodem als hulpbron is er behoefte aan een regelmatige bepaling van de chemische en fysische eigenschappen, zowel in ruimte als tijd. Kwantitatieve schatting van de exacte hoeveelheid, ruimtelijke verdeling en temporele verandering van bodemeigenschappen is nog steeds een uitdaging. Het onderwerp van dit proefschrift is hoe spectrale reflectie informatie gelinkt kan worden aan bodemeigenschappen
The contributions of Dr. Alexander F.H. Goetz to imaging spectrometry
MacDonald, J.S. ; Ustin, S.L. ; Schaepman, M.E. - \ 2009
Remote Sensing of Environment 113 (2009)Suppl.1. - ISSN 0034-4257 - p. S2 - S4.
aviris - spectroscopy - earth
Hyperspectral remote sensing is the definitive optical tool for increasing knowledge and understanding of the Earth's surface. Contiguous high-resolution spectrometry provides a new dimension in mapping capability because of the potential for quantitative measurement of surface biogeochemistry. Alexander Goetz provided the vision and leadership that has produced nearly all critical developments in this field. He was among the first to recognize that spectrometry would change optical remote sensing from qualitative observations to quantitative physical measurements. His significant accomplishments over the last 25 years include development of critical image processing and atmospheric correction software, spectrometers that made it possible to move research out of the lab and into the field environment, and the development of NASA's airborne imaging spectrometer program. This special issue is dedicated to Dr. Goetz and his accomplishments
Structural properties of a peptide derived from H+-V-ATPase subunit a
Vermeer, L.S. ; Reat, V. ; Hemminga, M.A. ; Milon, A. - \ 2009
Biochimica et Biophysica Acta. Biomembranes 1788 (2009)5. - ISSN 0005-2736 - p. 1204 - 1212.
proton translocation channel - mediated cross-linking - vacuolar (h+)-atpases - transmembrane segments - magnetic-resonance - nmr-spectra - topology - domain - spectroscopy - surfaces
The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H+-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit
Membrane protein frustration: protein incorporation into hydrophobic mismatched binary lipid mixtures
Stopar, D. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Biophysical Journal 96 (2009)4. - ISSN 0006-3495 - p. 1408 - 1414.
major coat protein - bacteriophage m13 - phase-transitions - acyl-chain - bilayers - fluid - phosphatidylcholines - spectroscopy - domain - solubilization
Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall ¿-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified
Adsorption of Anionic Surfactants in a Nonionic Polymer Brush: Experiments, Comparison with Mean-Field Theory, and Implications for Brush-Particle Interaction
Vos, W.M. de; Biesheuvel, P.M. ; Keizer, A. de; Kleijn, J.M. ; Cohen Stuart, M.A. - \ 2009
Langmuir 25 (2009)16. - ISSN 0743-7463 - p. 9252 - 9261.
size-exclusion chromatography - sodium dodecyl-sulfate - aqueous-solutions - grafted polymers - hard-spheres - reflectometry - spectroscopy - monolayers - scattering - surfaces
The adsorption of the anionic surfactants sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS) in poly(ethylene oxide) (PEO) brushes was studied using a fixed-angle optical flow-cell reflectometer. We show that, just as in solution, there is a critical association concentration (CAC) for the surfactants at which adsorption in the PEO brush starts. Above the critical micelle concentration (CMC) the adsorption is found to be completely reversible. At low brush density the adsorption per PEO monomer is equal to the adsorption of these surfactants in bulk solution. However, with increasing brush density, the number of adsorbed surfactant molecules per PEO monomer decreases rapidly. This decrease is explained in terms of excluded volume interactions plus electrostatic repulsion between the negatively charged surfactant micelles. Experimentally, a plateau value in the total adsorption is observed as a function of grafting density. The experimental results were compared to the results of an analytical self-consistent field (aSCF) model, and we found quantitative agreement. Additionally, the model predicts that the plateau value found is in fact a maximum. Both experiments and model calculations show that the adsorption scales directly with the polymerization degree of the polymers in the brush. They also show that an increase in the ionic strength leads to an increase in the adsorbed amount, which is explained as being due to a decrease in the electrostatic penalty for the adsorption of the SDS micelles. The adsorption of SDS micelles changes the interactions of the PEO brush with a silica particle. This is illustrated by atomic force microscopy (AFM) measurements of the pull-off force of a silica particle from a PEO brush: at high enough PEO densities, the addition of SDS leads to a very strong reduction in the force necessary to detach the colloidal silica particle from the PEO brush. We attribute this effect to the large amount of negative charge incorporated in the PEO brush due to SDS adsorption
Primary photosynthetic processes: from supercomplex to leaf
Broess, K. - \ 2009
Wageningen University. Promotor(en): Herbert van Amerongen. - [S.l.] : S.n. - ISBN 9789085852988 - 124
fotosynthese - fluorescentie - fluorescentiemicroscopie - spectroscopie - membranen - chloroplasten - fotosysteem ii - planten - photosynthesis - fluorescence - fluorescence microscopy - spectroscopy - membranes - chloroplasts - photosystem ii - plants
This thesis describes fluorescence spectroscopy experiments on photosynthetic complexes that cover the primary photosynthetic processes, from the absorption of light by photosynthetic pigments to a charge separation (CS) in the reaction center (RC). Fluorescence spectroscopy is a useful tool in photosynthetic particles, because the latter are densely packed with fluorescence pigments like chlorophylls (Chl). The fluorescence of each pigment is affected by its environment and provide information about structure and dynamics of the photosynthetic complexes. In this thesis time-resolved fluorescence of Chl molecules is used for studying the ultrafast kinetics in membrane particles of photosystem II (PSII) (chapter 2, 3 and 4). In chapter 5 fluorescence lifetime imaging microscopy (FLIM) of is applied to study entire chloroplasts, either in the leaf or in isolated chloroplast form. The advantage of FLIM is that the interactions of the fluorescence pigments in both photosystems can be spatially resolved up to a resolution of 0.5 x 0.5 x 2 µm to indentify and quantify photosynthetic processes in their natural environment.
Excitation energy transfer and charge separation in PSII membranes (chapter 2,3 and 4)
In this thesis time-resolved fluorescence measurements of PSII containing membranes, the so called BBY particles, are performed in low-light conditions with open reaction centers. The BBY particles do not contain photosystem I (PSI) or stroma lamellae, but do support electron transfer and carry out oxygen evolution with high activity and are comparable with the grana in vivo. The fluorescence decay kinetics of the BBY particles are faster than observed in previous studies and also faster than observed for PSII in chloroplasts and thylakoid preparations. The average lifetime is 150 ps, which, together with previous annihilation experiments on light-harvesting complex II (LHCII) suggests that excitation migration from the antenna complexes contributes significantly to the overall charge separation time. This is in disagreement with the commonly applied exciton / radical-pair-equilibrium (ERPE) model that assumes that excitation energy diffusion through the antenna to the RC is much faster than the overall charge-separation time.
A simple coarse-grained method is proposed, based on the supramolecular organization of PSII and LHCII in grana membranes (C2S2M2). The proposed modelling procedure for BBY particles is only approximate and many different combinations of excitation migration time and the charge separation time can explain the observed fluorescence kinetics. However it is clear that charge transfer should be rather fast and is accompanied with a large drop in free energy.
In chapter 3, the fluorescence kinetics of BBY particles with open RCs are compared after preferential excitation at 420 and 484 nm, which causes a difference in the initial excited-state populations of the inner and outer antenna system. The fluorescence decay is somewhat slower upon preferential excitation of chlorophyll (Chl) b, which is exclusively present in the outer antenna. Using the coarse-grained model it was possible to fit the 420 and 484 nm results simultaneously with a two-step electron transfer model and four parameters: the hopping rate between the protein-pigment complexes, the CS rate, the drop in free energy upon primary charge separation and a secondary charge separation rate. The conclusion is that the average migration time contributes ~25% to the overall trapping time. The hopping time obtained in chapter 3 is significantly faster than might be expected based on studies on trimeric and aggregated LHCII and it is concluded that excitation energy transfer in PSII follows specific pathways that require an optimized organization of the antenna complexes with respect to each other. Analysis of the composition of the BBY particles indicates that the size of the light-harvesting system in PSII is smaller than commonly found for PSII in chloroplasts and explains why the fluorescence lifetimes are smaller for the BBY’s.
In chapter 4, four different PSII supercomplex preparations were studied. The main difference between these supercomplexes concerns the size of the outer antenna. The average lifetime of the supercomplexes becomes longer upon increasing the antenna size. The results indicate that the rate constants obtained from the coarse-grained method for BBY preparations, which is based on the supercomplex composition C2S2M2, should be slightly faster (~10%) as predicted in chapter 3. The observation that the average lifetime of the supercomplexes is relatively slow compared to what one might expect based on the measurements on BBY particles, and this will require further future studies.
Photosynthesis in plant leaves (Chapter 5)
With the use of femtosecond two-photon excitation TPE at 860 nm it appears to be possible to measure fluorescence lifetimes throughout the entire leaves of Arabidopsis thaliana and Alocasia wentii. It turns out that the excitation intensity can be kept sufficiently low to avoid artifacts due to singlet-singlet and singlet-triplet annihilation, while the reaction centers can be kept in the open state during the measurements. The average fluorescence lifetimes obtained for individual chloroplasts of Arabidopsis thaliana and Alocasia wentii in the open and closed state, are approximately ~250 ps and ~1.5 ns, respectively. The maximum fluorescence state correspond to a state in which all reaction centers are closed. The kinetics are very similar to those obtained for chloroplasts in vitro with the FLIM setup and to in vivo results reported in literature. No variations between chloroplasts are observed when scanning throughout the leaves of Arabidopsis thaliana and Alocasia wentii. Within individual chloroplasts some variation is detected for the relative contributions of PSI and PSII to the fluorescence. The results open up the possibility to use FLIM for the in vivo study of the primary processes of photosynthesis at the level of single chloroplasts under all kinds of (stress) conditions.
This thesis gives new insight of the kinetic processes in PSII membranes. With the use of a coarse-grained method that provides an easy way to incorporate existing knowledge and models for individual complexes, valuable conclusions can be drawn about the excitation energy transfer and the CS which hopefully contributes to an improvement of the knowledge about PSII functioning. In general it was shown that a large drop in free energy is needed in PSII membranes for all simulations with the coarse-grained method.
The presented results on the kinetics of chloroplasts obtained in vitro and in vitro are very similar and verify that conclusions drawn from isolated chloroplasts can be extrapolated to photosynthetic processes in their natural environment.
Increased susceptibility of ß-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures
Bruins, M.E. ; Meersman, F. ; Janssen, A.E.M. ; Heremans, K. ; Boom, R.M. - \ 2009
FEBS Journal 276 (2009). - ISSN 1742-464X - p. 109 - 117.
enzyme inactivation - escherichia-coli - proteins - stabilization - temperature - myoglobin - thermostability - denaturation - spectroscopy - aggregation
The stability of ß-glucosidase from the hyperthermophile Pyrococcus furiosus was studied as a function of pressure, temperature and pH. The conformational stability was monitored using FTIR spectroscopy, and the functional enzyme stability was monitored by inactivation studies. The enzyme proved to be highly piezostable and thermostable, with an unfolding pressure of 800 MPa at 85 °C. The tentative pressure¿temperature stability diagram indicates that this enzyme is stabilized against thermal unfolding at low pressures. The activity measurements showed a two-step inactivation mechanism due to pressure that was most pronounced at lower temperatures. The first part of this inactivation took place at pressures below 300 MPa and was not visible as a conformational transition. The second transition in activity was concomitant with the conformational transition. An increase in pH from 5.5 to 6.5 was found to have a stabilizing effect
8000 yr of black carbon accumulation in a colluvial soil from NW Spain
Kaal, J. ; Martinez-Cortizas, A. ; Buurman, P. ; Criado Boado, F. - \ 2008
Quaternary Research 69 (2008)1. - ISSN 0033-5894 - p. 56 - 61.
solid-state c-13 - organic-matter - chemical-composition - humic acids - fractions - pyrolysis - spectroscopy - spectrometry - cambisol - fire
Analytical pyrolysis-GC/MS and solid-state 13C NMR (nuclear magnetic resonance) were applied to the NaOH-extractable organic matter fraction of a colluvial soil from Galicia (NW Spain) that represents more than 8500 yr of accumulation. While molecular indicators of vegetation change were looked for, it seemed likely that any such signal was disturbed by the intense fire regime of the area. This conclusion was drawn from (1) the presence of three charcoal layers, (2) the high proportion of aryl C in NMR spectra (non-quantitative) and (3) the prevalence of benzenes and polycyclic aromatic hydrocarbons (PAHs) in the chromatograms (38 ± 6% of total identified peak area), also in charcoal-poor samples. If this conclusion is accurate, the area has been subjected to burning episodes for at least 8000 yr. Additionally, the results indicate that biomass burning residues (black carbon; BC) may become NaOH extractable after long periods of degradation in mineral soil. These results add to our knowledge of the long-term fate of BC in soil, which is a potential agent in the global C cycle.
Mechanism of endocarp-imposed constraints of germination of Lannea microcarpa seeds
Neya, O. ; Hoekstra, F.A. ; Golovina, E.A. - \ 2008
Seed Science Research 18 (2008)01. - ISSN 0960-2585 - p. 13 - 24.
rhus species anacardiaceae - physical dormancy - ftir microspectroscopy - desiccation tolerance - ethylene - spectroscopy - metabolism - embryos - sativa
Lannea microcarpa, a multipurpose tree species from the dry African savanna, sheds seeds that often display inhibition of germination. The underlying mechanism was investigated using seeds processed from fully matured fruits collected from natural stands in Burkina Faso. Germination of fresh seeds was variable (16¿28%), while they did not germinate after drying and rehydration. Mechanical scarification of the endocarp at the proximal end of the seeds increased germination to 83¿94%. Scarification on the distal end led to delayed radicle emergence through the produced hole in c. 40% of the seeds. The endocarp was permeable to water and respiratory gases. Increased water content in scarified seeds was associated with radicle extension during germination. Intact and scarified non-germinated seeds displayed a moderate rate of respiration with respiratory quotient (RQ) values of c. 1. Respiration increased and RQ decreased to c. 0.7 with radicle emergence. Ethylene evolution peaked in both intact and scarified seeds at the beginning of incubation and then decreased to low values. Inhibition of ethylene production by 1¿5 mM 2-amino-ethoxyvinylglycine (AVG) caused only a partial decrease of germination of the scarified seeds. Intact non-germinated seeds gradually lost viability during incubation at 30°C, but could be rescued by delayed scarification before day 15 of incubation. It is concluded that radicle emergence in dry L. microcarpa seeds is inhibited only mechanically. The mechanical properties of the endocarp are attributed to irreversible structural changes of the lignin¿hemicellulose complex, which occur during drying.
Adsorption of the protein bovine serum albumin in a planar poly(acrylic acid) brush layer as measured by optical reflectometry
Vos, W.M. de; Biesheuvel, P.M. ; Keizer, A. de; Kleijn, J.M. ; Cohen Stuart, M.A. - \ 2008
Langmuir 24 (2008)13. - ISSN 0743-7463 - p. 6575 - 6584.
spherical polyelectrolyte brushes - electrostatic interactions - charge regulation - aqueous-solution - complexes - spectroscopy - chloride) - particles
The adsorption of bovine serum albumin (BSA) in a planar poly(acrylic acid) (PAA) brush layer has been studied by fixed-angle optical reflectometry. The influence of polymer length, grafting density, and salt concentration is studied as a function of pH. The results are compared with predictions of an analytical polyelectrolyte brush model, which incorporates charge regulation and excluded volume interactions. A maximum in adsorption is found near the point of zero charge (pzc) of the protein. At the maximum, BSA accumulates in a PAA brush to at least 30 vol %. Substantial adsorption continues above the pzc, that is, in the pH range where a net negatively charged protein adsorbs into a negatively charged brush layer, up to a critical pH value. This critical pH value decreases with increasing ionic strength. The adsorbed amount increases strongly with both increasing PAA chain length and increasing grafting density. Experimental data compare well with the analytical model without having to include a nonhomogeneous charge distribution on the protein surface. Instead, charge regulation, which implies that the protein adjusts its charge due to the negative electrostatic potential in the brush, plays an important role in the interpretation of the adsorbed amounts. Together with nonelectrostatic interactions, it explains the significant protein adsorption above the pzc.
Retrieval of chlorophyll concentration from leaf reflectance spectra using wavelet analysis
Blackburn, G.A. ; Ferwerda, J.G. - \ 2008
Remote Sensing of Environment 112 (2008)4. - ISSN 0034-4257 - p. 1614 - 1632.
hyperspectral data - vegetation - calibration - prospect - pigment - spectroscopy - indexes - models - red
The dynamics of foliar chlorophyll concentrations have considerable significance for plant¿environment interactions, ecosystem functioning and crop growth. Hyperspectral remote sensing has a valuable role in the monitoring of such dynamics. This study focussed upon improving the accuracy of chlorophyll quantification by applying wavelet analysis to reflectance spectra. Leaf-scale radiative transfer models were used to generate very large spectral data sets with which to develop and rigorously test refinements to the approach and compare it with existing spectral indices. The results demonstrated that by decomposing leaf spectra, the resultant wavelet coefficients can be used to generate accurate predictions of chlorophyll concentration, despite wide variations in the range of other biochemical and biophysical factors that influence leaf reflectance. Wavelet analysis outperformed predictive models based on untransformed spectra and a range of spectral indices. The paper discusses the possibilities for further refining the wavelet approach and for extending the technique to the sensing of a variety of vegetation properties at a range of spatial scales.
Water content of acacia honey dertermined by two established methods and by optothermal window
Szopos, S. ; Doka, O. ; Bicanic, D.D. ; Ajtony, Z. - \ 2008
Acta Chimica Slovenica 55 (2008)2. - ISSN 1318-0207 - p. 273 - 276.
The major objective of the research study described here was to explore the potential of the optothermal window (OW) technique as a new approach towards a simple, rapid determination of water content in honey. Water, major component of foods, influences their physical and chemical properties. Single mode RLT-1480-40G laser diode and the standard addition method were used to calibrate the response of the OW detector at analytical wavelength of 1478 nm and to determine water content of Acacia honey. The performance of the OW method was compared to that of well established gravimetry and refractometry; the values obtained by the three different methods are practically the same
Practical, reliable and inexpensive assay of lycopene in tomato products based on the combined use of light emitting diode (LED) and the optothermal window
Bicanic, D.D. ; Cuypers, R. ; Luterotti, S. ; Sporec, M. ; Zoppi, A. ; Vugec, J. - \ 2008
Acta Chimica Slovenica 55 (2008)2. - ISSN 1318-0207 - p. 468 - 473.
performance liquid-chromatography - quantification - spectroscopy
Light emitting diode (LED) combined with the concept of optothermal window (OW) is proposed as a new approach (LED-OW) to detect lycopene in a wide range of tomato-based products (tomato juice, tomato ketchup, tomato passata and tomato puree). Phytonutrient lycopene is a dominant antioxidant in these products while beta-carotene is present in significantly lower quantities. Therefore for all practical reasons the interfering effect of beta-carotene at 502 nm analytical wavelength can be neglected. The LED-OW method is low-cost and simple, yet accurate and precise. The major attributes of the new method are its rapid speed of response and the fact that no preparation whatsoever of the sample is needed before the analysis. The lycopene found in tomato products studied here varies from 8 mg/100 g to 60 mg/100 g fresh product. Results obtained by LED-OW method were compared to the outcome of conventional, time consuming spectrophotometric methods and the correlation was very good (R = 0.98). Precision of the LED-OW instrumental setup ranged from 0.5 to 7.4%; the RSD achieved for lycopene-richest samples (= 40 mg/100 g) did not exceed 1.7%. Repeatability of analysis by LED-OW was found to vary between 0.7 and 7.1%.
Assessment of near infrared and "software sensor" for biomass monitoring and control
Soons, Z.I.T.A. ; Streefland, M. ; Straten, G. van; Boxtel, A.J.B. van - \ 2008
Chemometrics and Intelligent Laboratory Systems 94 (2008)2. - ISSN 0169-7439 - p. 166 - 174.
analytical technology pat - bordetella-pertussis - fermentation processes - growth-rate - calibration - design - online - chemometrics - spectroscopy - toxin
Spectroscopic instrumentation is often seen as promising for process analytical technology (PAT) to enhance control of manufacturing (bio)pharmaceuticals. The interpretation of near infrared spectra is challenging due to the large number of wavelengths recorded and the overlapping absorbance features of near infrared spectroscopy. This work applies a controlled random search procedure to select an optimal window of wavelengths giving a good calibration model for biomass concentrations during cultivation of Bordetella pertussis, the causative agent of whooping cough. The proposed wavelengths selection procedure outperforms the traditional calibration procedures. In the second half of the paper, the near infrared based predictions are compared with the estimations obtained from a software sensor for biomass and specific growth rate based in standard measurements of oxygen consumption. Both methods estimate the exponential biomass growth properly. The near infrared predictions depend on the quality of the training dataset, which needs to encompass all possible sources of temporary disturbances like pH and dissolved oxygen. If the training dataset does not comprise such disturbances, then the accuracy and robustness of the near infrared predictions are less favorable than those of the software sensor. Although near infrared has the potential to provide more information than just biomass, the software sensor is the preferred choice for feedback control of biomass and specific growth rate
Alkyl-Functionalized Oxide-Free Silicon Nanoparticles: Synthesis and Optical Properties
Rosso-Vasic, M. ; Spruijt, E. ; Lagen, B. van; Cola, L. de; Zuilhof, H. - \ 2008
Small 4 (2008)10. - ISSN 1613-6810 - p. 1835 - 1841.
hydrogen-terminated silicon - porous silicon - surface functionalization - nanocrystals - hydrosilylation - monolayers - luminescence - spectroscopy - photoluminescence - nanoclusters
Highly monodisperse silicon nanoparticles (1.57 ± 0.21 nm) are synthesized with a covalently attached alkyl monolayer on a gram scale. Infrared spectroscopy shows that these silicon nanoparticles contain only a few oxygen atoms per nanoparticle. XPS spectra clearly show the presence of unoxidized Si and attached alkyl chains. Owing to the relatively efficient synthesis (yields 100-fold higher than of those previously reported) the molar extinction coefficient can be measured: max = 1.7 × 10-4 M-1cm-1, only a factor of 4 lower than that of CdS and CdSe nanoparticles of that size. The quantum yield of emission ranges from 0.12 (C10H21-capping) to 0.23 (C16H33-capping). UV/Vis absorption and emission spectroscopy show clear vibrational progressions (974 ± 14 cm-1; up to five vibrational bands visible at room temperature), resembling bulk SiC phonons, which support the monodispersity observed by TEM. This was also confirmed by time-resolved fluorescence anisotropy measurements, which display a strictly monoexponential decay that can only be indicative of monodisperse, ball-shaped nanoparticles
Physical interactions among plant MADS-box transcription factors and their biological relevance
Nougalli Tonaco, I.A. - \ 2008
Wageningen University. Promotor(en): Sacco de Vries; Gerco Angenent, co-promotor(en): Richard Immink. - [S.l.] : S.n. - ISBN 9789085048299 - 150
planten - transcriptiefactoren - genexpressie - transcriptie - petunia hybrida - arabidopsis thaliana - fluorescentie - spectroscopie - dna-bindende eiwitten - bloemen - technieken - plantenontwikkeling - genregulatie - transcriptieregulatie - plants - transcription factors - gene expression - transcription - petunia hybrida - arabidopsis thaliana - fluorescence - spectroscopy - dna binding proteins - flowers - techniques - plant development - gene regulation - regulation of transcription
The biological interpretation of the genome starts from transcription, and many different signaling pathways are integrated at this level. Transcription factors play a central role in the transcription process, because they select the down-stream genes and determine their spatial and temporal expression. In higher eudicot species around 2000 specific transcription factors are present, which can be classified into families based on conserved common domains. The MADS-box transcription factor family is an important family of transcription regulators in plants and genetic studies revealed that members of this family are involved in various developmental processes, like floral induction, floral organ formation and fruit development. In contrast to this wealth of information concerning MADS-box gene functions, the molecular mode of action of the encoded proteins is far from completely understood. Biochemical and yeast η-hybrid experiments performed in the past showed that MADS-box proteins are able to interact mutually, and based on these findings a hypothetical quaternary model has been proposed as molecular working mechanism. According to this model two MADS-box protein dimers assemble into a higher order complex, which binds DNA and regulates target gene expression. Although, this molecular mechanism sounds plausible, it still lacks evidence from in vivo studies. In this study we investigated physical interactions among members of the Petunia hybrida and Arabidopsis thaliana MADS-box transcription factor families in living plant cells. For this purpose, sophisticated micro-spectroscopy techniques have been implemented and in addition, some novel fluorescent-protein-based tools were developed. The first chapter gives an introduction about the dynamic transcriptional process and describes our current knowledge about transcriptional regulation in eukaryotes. The central question of this chapter is how transcription factors are able to find their specific binding sites (ci's-elements) within the huge genome. The various mechanisms, such as "looping" and "sliding", that have been proposed are discussed, as well as the relevance of direct interactions between transcription factors for the control of gene expression.
In a first attempt to detect protein interactions in living cells, we transiently expressed combinations of petunia MADS-box transcription factors labeled with different color variants of the Green Fluorescent Protein (GFP) in leaf protoplasts (Chapter 2). Subsequently, the transfected protoplasts were analyzed by means of FRET-FLIM
(Fluorescence Resonance Energy Transfer - Fluorescence Lifetime Imaging) to identify specific dimerization. In addition, we have obtained indirect evidence for higher-order complex formation of the petunia MADS-box proteins FLORAL BINDING PROTEIN2 (FBP2), FBP11, and FBP24 in living cells. Similar kind of analyses for Arabidopsis MADS-box proteins involved in petal and stamen development revealed clear differences in interaction affinities in vivo and furthermore, many homodimers were identified that could not be detected by yeast-based systems in the past (Chapter 3). This result demonstrated the robustness of the FRET-FLIM approach. Based on our observations, we hypothesize that 'partner selectivity' plays an important role in complex formation at particular developmental stages. To study differences in interaction affinity and selectivity and the consequences for complex formation in more detail, a novel method was developed (Chapter 4). The technique, designated "Competition-FRET", allows the verification of competition effects between proteins, and furthermore, it may provide information about the formation of higher-order complexes between different proteins under study. The developed method was implemented to investigate in depth the preference for homo- or heterodimer interactions of the Arabidopsis MADS-box proteins AGAMOUS (AG) and SEPALLATA3 (SEP3).
The detection of interactions in living cells by FRET as it has been done in the studies described above demands a sophisticated microscopy set-up, and therefore, we decided to test and implement an alternative and theoretically simple technique (Chapter 5). This method for the in vivo detection of protein-protein interaction is called BiFC (Bimolecular Fluorescence Complementation), or "Split-YFP". In this system, a fluorescent molecule is split into two inactive domains and these two non-fluorescent parts are fused to the proteins under study. Only upon interaction of the two protein partners the two non-fluorescent parts of the fluorescent molecule are brought into close proximity, which enables the recovery of fluorescence. We used the EYFP (Enhanced Yellow Fluorescence Protein) molecule as fluorescent molecule and were able to detect the interaction between AG and SEP3 in nuclei of Arabidopsis leaf protoplasts. Techniques like this and FRET-FLIM allow the analyses of interactions between proteins in living cells, but give no information about the size of the formed complexes. To get a first indication about the stoichiometry of protein complexes, we monitored the diffusion time of in vitro synthesized AG-EYFP and SEP3-EYFP fusion proteins by means of FCS (Fluorescence Correlation
Spectroscopy). From these experiments described in Chapter 6, we could speculate that SEP3 is present as a dimer and also as a higher order complex, whilst AG on its own is able to assemble into larger complexes. The diffusion time of the product formed upon co-translation of both AG and SEP3, suggests that a multimenc protein complex with a high molecular weight is formed upon interaction between AG and SEP3. Even though FCS is a powerful technique, these interpretations should be taken cautiously, mainly because these experiments were done in vitro instead of in living cells. Finally, in the last chapter we discuss the various methods that have been implemented and developed to monitor protein-protein interactions and complex formation of MADS-box transcription factors in living plant cells. Furthermore, we made a first step to monitor interactions in intact tissues under endogenous expression levels, and the preliminary results obtained from these in planta FRET-FLIM measurements are discussed.
Imaging spectroscopy : applications in agriculture
Zedde, H.J. van de; Brakel, R.P. van - \ 2007
spectroscopie - kwaliteitscontroles - monitoring - sensorische evaluatie - voedselinspectie - tomaten - patates frites - beeldvormende spectroscopie - spectroscopy - quality controls - monitoring - sensory evaluation - food inspection - tomatoes - chips (French fries) - imaging spectroscopy
Imaging Spectroscopy is the study of light as a function of spatial distribution and wavelength that has been transmitted, emitted, reflected or scattered from an object. This allows us to derive information about the spatial relation of the chemistry of the object. Imaging spectroscopy is suited for the following tasks: • Quality control: detection of latent defects in agri-products, e.g. vegetables and fruit. • Quantification of compounds: carotenes, proteins, sugars, moisture etc. In this poster the following two applications are discussed: 1) Measuring of compounds in tomatoes and 2) Detection and classification of latent defects in French Fries
Negative compressibility and non-equivalence of two statistical ensembles in the escape transition of a polymer chain
Skvortsov, A.M. ; Klushin, L.I. ; Leermakers, F.A.M. - \ 2007
Journal of Chemical Physics 126 (2007). - ISSN 0021-9606 - p. 024095 - 024095.
atomic-force microscopy - spectroscopy - elasticity - surface
An end-tethered polymer chain compressed between two pistons undergoes an abrupt transition from a confined coil state to an inhomogeneous flowerlike conformation partially escaped from the gap. This phase transition is first order in the thermodynamic limit of infinitely long chains. A rigorous analytical theory is presented for a Gaussian chain in two ensembles: (a) the H-ensemble, in which the distance H between the pistons plays the role of the independent control parameter, and (b) the conjugate f-ensemble, in which the external compression force f is the independent parameter. Details about the metastable chain configurations are analyzed by introducing the Landau free energy as a function of the chain stretching order parameter. The binodal and spinodal lines, as well as the barrier heights between the stable and metastable states in the free energy landscape, are presented in both ensembles. In the loop region for the average force with dependence on the distance H (i.e., in the H-ensemble) a negative compressibility exists, whereas in the f-ensemble the average distance as a function of the force is strictly monotonic. The average fraction of imprisoned segments and the lateral force, taken as functions of the distance H or the average H, respectively, have different behaviors in the two ensembles. These results demonstrate a clear counterexample of a main principle of statistical mechanics, stating that all ensembles are equivalent in the thermodynamic limit. The authors show that the negative compressibility in the escape transition is a purely equilibrium result and analyze in detail the origin of the nonequivalence of the ensembles. It is argued that it should be possible to employ the escape transition and its anomalous behavior in macroscopically homogeneous, but microscopically inhomogeneous, materials.
FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein
Nazarov, P.V. ; Koehorst, R.B.M. ; Vos, W.L. ; Apanasovich, V.V. ; Hemminga, M.A. - \ 2007
Biophysical Journal 92 (2007)4. - ISSN 0006-3495 - p. 1296 - 1305.
bacteriophage m13 - transmembrane domain - energy-transfer - fluorescence - dynamics - conformation - spectroscopy - modulation - peptides - micelles
A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall -helical protein conformation from amino acid residues 12¿46, close to the protein conformation in the intact phage.
Evaluating pyrolysis-GC/MS and 13C CPMAS NMR in conjunction with a molecular mixing model of the Penido Vello peat deposit, NW Spain
Kaal, J. ; Baldock, J.A. ; Buurman, P. ; Nierop, K.G.J. ; Pontevedra-Pombal, X. ; Martínez-Cortizas, A. - \ 2007
Organic Geochemistry 38 (2007)7. - ISSN 0146-6380 - p. 1097 - 1111.
soil organic-matter - polycyclic aromatic-hydrocarbons - ionization cross-sections - mass-spectrometry - state - spectroscopy - acids - alkylbenzenes - preservation - fractions
We performed solid state 13C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectroscopy and pyrolysis¿gas chromatography/mass spectrometry (Py¿GC/MS) on the Penido Vello peat deposit located in Galicia, NW Spain. Often regarded as complementary techniques, solid state 13C NMR and Py¿GC/MS are widely used for the characterisation of organic matter. Recently, a molecular mixing model (MMM) was proposed to predict the distribution of C in biochemical components (carbohydrate, protein, lignin, lipid, char) from the 13C NMR spectral distribution, thereby allowing a quantitative comparison with Py¿GC/MS product abundances. We discuss the application of this model to a peat core, by comparing NMR-MMM results with Py¿GC/MS data. The core represents 5000 yr accumulation and ranges from fibric (at the surface) to hemic (bottom). The amounts of carbohydrates and lipids predicted by the MMM and calculated from the quantified Py¿GC/MS chromatograms are in close agreement. However, the well known low capability of the conventional Py¿GC/MS method to provide structural information on proteins from bulk soils, the poor GC amenability of polar compounds and many other possible sources of inaccuracy in Py¿GC/MS and NMR-MMM caused discrepancies with predictions made with the MMM. Also, the MMM failed to give good prediction of the C/N ratio of the peat material. Although the NMR-MMM approach does not account for molecular transitions during decomposition, this oversimplification proved to be acceptable. The MMM seems to be a useful tool for the interpretation of NMR spectral distributions in peat material.
Determination of the degree of substitution, degree of amidation and degree of blockiness of commercial pectins by using capillary electrophoresis
Guillotin, S.E. ; Bakx, E.J. ; Boulenguer, P. ; Schols, H.A. ; Voragen, A.G.J. - \ 2007
Food Hydrocolloids 21 (2007)3. - ISSN 0268-005X - p. 444 - 451.
galacturonic acid distribution - de-esterified pectins - zone-electrophoresis - quantification - endopolygalacturonase - spectroscopy - separation - behavior
It is more and more realised that pectins are complex mixtures of many different molecules and research is directed towards the fractionation and characterisation of these pectic sub-populations. Since fractionation of pectins generally results in only low amounts of purified material, rapid characterisation methods using low amounts of samples are required. In this study, capillary electrophoresis was chosen to characterise pectins because only tiny amounts of sample are needed for the analysis. A new capillary electrophoresis (CE) protocol was developed to determine the degree of amidation, the degree of methyl-esterification (DM) and consequently the degree of substitution (DS) of pectins by analysing the pectins before and after removal of the methyl-esters. The CE results were compared with results obtained for the same pectins by using titration and Fourier transform infra-red (FTIR) spectroscopy methods. The CE method was found to be rather reliable resulting in small standard deviations for the DS and DAm. The CE method had the advantage of being rapid due to the limited sample preparation and automation of the analysis. In addition, CE was used successfully to determine the degree of blockiness of the free GalA residues over the pectic backbone.
Red edge shift and biochemical content in grass canopies
Mutanga, O. ; Skidmore, A.K. - \ 2007
ISPRS Journal of Photogrammetry and Remote Sensing 62 (2007)2. - ISSN 0924-2716 - p. 34 - 42.
kruger-national-park - vegetation indexes - absorption features - chlorophyll content - leaf reflectance - south-africa - nitrogen - spectroscopy - regression - quality
The concentration of foliar nitrogen in tropical grass is one of the factors that explain the distribution of wildlife. Therefore, the remote sensing of foliar nitrogen contributes to a better understanding of wildlife feeding patterns. This study evaluated changes in the red edge position of the 680 nm continuum removed chlorophyll feature in the reflectance spectra of samples of Cenchus ciliaris grass grown in a greenhouse under three levels of nitrogen supply. Canopy spectral measurements from each treatment were recorded under controlled laboratory conditions over a four-week period using a GER 3700 spectroradiometer. Results indicate that the mean wavelength positions of the three fertilization treatments were statistically different. An increase in nitrogen supply yielded a shift in the red edge position to longer wavelengths. The red edge position, amplitude, slope at 713 nm and slope at 725 nm were significantly correlated to measured nitrogen concentration (bootstrapped r=0.89, -0.28, 0.63 and 0.75, respectively) even at canopy level. Based on these results, the red edge position is strongly correlated with biochemical concentration in plants compared to the other methods tested. The study provides conclusive evidence that confirms the strength of a red edge-nitrogen relationship that remains underused in remote sensing. This method is promising for estimating nutrient content in grasslands. (C) 2007 International Society for Photogrammetry and Remote Sensing, Inc. (ISPRS). Published by Elsevier B.V. All rights reserved.
Structure-rheology relations in sodium caseinate containing systems
Ruis, H.G.M. - \ 2007
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Paul Venema. - [S.l.] : S.n. - ISBN 9789085046486 - 125
natriumcaseïnaat - reologische eigenschappen - afschuifkracht - gelering - emulsies - structuur - verzuring - spectroscopie - licht - verstrooiing - sodium caseinate - rheological properties - shear - gelation - emulsions - structure - acidification - spectroscopy - light - scattering
The general aim of the work described in this thesis was to investigate structure-rheologyrelations for dairy related products, focusing on model systems containing sodium caseinate. The acid inducedgelationof sodium caseinate, of sodium caseinate stabilized emulsions, and the effect of shear on the structure formation was characterized. Special attention was given to the sol-gel transition point, which was defined by a frequency independent loss tangent. It was shown that the sol-gel transition point is completely controlled by the pH and the temperature, independent of the concentration sodium caseinate or the applied shear rate. Considering sodium caseinate solutions, increase of the temperature of acidification caused a decrease of the critical pH forgelationand a more dense gel structure. The formed gels were not in thermodynamicequilibrium,however, due to the slow kinetics of the system they were stable on the time scale of the experiment. At the gel point we have strong indications that the structure can not be characterized by a single fractal dimension. During the acid inducedgelationof sodium caseinate stabilized emulsions a single sol-gel transition was observed. Addition of an excess of sodium caseinate to the emulsion resulted in two sol-gel transitions upon acidification. Application of shear during the acidification of the emulsions showed a decreasing radius of the aggregates formed at thegelpointwith increasing shear rate. The aggregates formed becamemore densedue to the application of shear while the network that was formed by the aggregates became less compact. No shear induced alignment was observed of emulsion droplets dispersed in water or ina sodiumcaseinatesolution, while emulsion droplets dispersed in axanthansolution did align in a shear field. Addition of sodium inhibited the string formation of the emulsion droplets
New developments in the detection and identification of processed animal proteins in feeds
Raamsdonk, L.W.D. van; Holst, C. von; Baeten, V. ; Berben, G. ; Boix, A. ; Jong, J. de - \ 2007
Animal Feed Science and Technology 133 (2007)1-2. - ISSN 0377-8401 - p. 63 - 83.
bovine spongiform encephalopathy - bone meal - monoclonal-antibody - mitochondrial-dna - mass-spectrometry - pcr detection - ruminant - spectroscopy - validation - dipeptides
It is generally accepted that the most likely route of infection of cattle with bovine spongiform encephalopathy (BSE) is by consumption of feeds containing low levels of processed animal proteins (PAPs). This likely route of infection resulted in feed bans, which were primarily aimed at ruminant feeds, and were later extended to all feeds for farmed animals. The feed bans were expected to develop into a future enforcement of the "species-to-species" ban, which prohibits only the feeding of animal-specific proteins to the same species. The species-to-species ban requires support of species-specific identification methods. In the European Union, microscopic evaluation is currently the only accepted method for the detection and characterization of PAPs in feeds, since it is possible to detect contaminations at the requested level of 1 g/kg with hardly any false negative nor positive results. This method is predominantly focused on the presence and characteristics of bone fragments, although other structures, e.g. muscle fibres, may provide circumstantial evidence of the respective animal types. Recent developments are the identification of bone fragments at the level of classes (mammal versus bird versus fish), supported by image analysis of bone characteristics. Detection of DNA and specific proteins are additional methods that can be applied for the identification of PAPs in feeds. DNA is known to be very specific for animal species and breeds, whereas proteins can also indicate the type of tissue. The latter aspect is important to differentiate between proteins that are authorised in animal nutrition from banned proteins. Improvements can be noted in recent years for both methods. For a proper application of polymerase chain reaction (PCR) to detect specific sequences of DNA, primer sets have been developed which amplify a DNA sequence shorter than approximately 100 nucleotides. Specific antibodies have been developed for protein detection of ruminant or bovine material. Recent results of various studies indicate that specific DNA and protein detection methods can detect PAPs at a contamination level of 1 g/kg. However, full validation of these methods still needs to be carried out. Other methods such as near-infrared spectroscopy (NIRS), near-infrared microscopy (NIRM), near-infrared imaging, liquid chromatography (LC) and olfactometry techniques can and will be applied for the detection of PAPs. NIRS is a non-destructive method that can be applied on-line in feed production plants. Generally, the detection limit is still too high to be applied in official control laboratories. Nevertheless, industrial application is feasible. NIRM and near-infrared imaging are techniques that allow collection of near-infrared spectra from individual particles. The level of detection is lower than 1 g/kg since it is based on the microscopic technique, in combination with the option of identification of the individual particles. LC is based on the detection and, if present, the ratio of different polypeptides. For example, carnosine is mainly present in mammals and anserine mainly found in birds. Olfactometry is based on detection of volatile non-specific agents. It is a non-destructive and fast technique. For both LC and olfactometry it appears that the presence of fish material masks the detection of proteins of land animals, even at a contamination level of 5 g/kg. Since 2003 five different proficiency studies and ring trials have been organized. The first proficiency study, allowing the participants to apply their own protocol, revealed that correct microscopic detection of 1 g/kg of mammalian PAP in the presence of 50 g fish meal/kg was realised in 0.44 of the cases. However, a bench mark study organized in the same year showed that a microscopic detection of 0.98 can be reached provided the application of an optimal protocol and a sufficient level of expertise. More recent studies showed that training, the application of a decision support system and use of an improved microscopy protocol resulted in a higher sensitivity. An attractive approach is the combination of the very low detection level of microscopy with identification by other methods. Several strategies for a combination of screening and confirmation methods are discussed in the present paper. The new developments in methodology will support current or new legislation (e.g. species-to-species ban, general application of fish meal). (c) 2006 Elsevier B.V. All rights reserved.
Distance constraints from site-directed spectroscopy as a tool to study membrane protein structure
Vos, W.L. - \ 2007
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga. - [S.l.] : S.n. - ISBN 9789085046257 - 106
oppervlakte-eiwitten - moleculaire structuur - spectroscopie - surface proteins - molecular conformation - spectroscopy
Membrane proteins are involved in nearly every process in the living cell. Their scientific importance cannot be overstated, and they account for nearly 60% of all prescribed drugs. Despite being an abundant and important class of proteins, high-resolution structural data on membrane proteins are relatively scarce. X-ray diffraction and NMR spectroscopy are routinely applied nowadays for the determination of structures of water-soluble proteins. However, for membrane proteins that require an amphipathic environment, there is not yet a well-defined strategy for obtaining the structure. For this reason, techniques based on site-directed labeling are being developed to study membrane proteins in their natural environment. In this work, we use two techniques based on the dipole-dipole interaction between two labels, electron spin resonance (ESR) and fluorescence (or Förster) resonance energy transfer (FRET) to obtain low-resolution (0.3-3 nm) distance information on the structure of membrane peptides. FRET is used to study the conformation of a reference membrane protein, i.e. M13 major coat protein, in fully hydrated vesicles. The FRET-derived distance constraints are used to refine the set of high-resolution structures that is available in the protein databank. We show that the coat protein adopts an extended conformation that is not very different from the conformation in the phage particle. In a separate part of this work, we use the FRET approach to monitor the conformation of the coat protein under conditions of hydrophobic mismatch. Although it was suggested that transmembrane protein domains can adapt their backbone conformation to different conditions of hydrophobic stress and that M13 coat protein is a flexible protein that can adapt to a multitude of environments, we show that the conformation of the coat protein in fact is similar under different conditions of hydrophobic mismatch. A parallel approach, based on ESR spin labeling, is used to study the conformation of a peptide that is derived from the crucial proton translocating domain of vacuolar ATPase. First we present a method to enhance the analysis for the determination of distances between two spin labels based on matrix-assisted laser desorption/ionization - time of flight mass spectrometry. Secondly, we use the data from the ESR experiments to study the structure of the peptide. Based on the combined results from the ESR experiments, molecular dynamics simulations and circular dichroism studies we conclude that the peptide forms a dynamica-helix when bound to SDS micelles. We discuss these findings in the light of the current models for proton translocation in the vacuolar ATPase.
MALDI-TOF MS evidence for the linking of flax bast fibre galactan to rhamnogalacturonan backbone
Gur'janov, O.P. ; Gorshkova, T.A. ; Kabel, M.A. ; Schols, H.A. ; Dam, J.E.G. van - \ 2007
Carbohydrate Polymers 67 (2007)1. - ISSN 0144-8617 - p. 86 - 96.
linum-usitatissimum l - cell - polysaccharides - spectroscopy - degradation - transition - oligomers - pectins - walls
Fibre-specific (1 ¿ 4)-ß-galactan extracted from bast fibre peels of developing flax (Linum usitatissimum L.) stem has been studied to elucidate its structural details. The polysaccharide was characterized by NMR and subjected to partial degradation protocols, including chemical and enzymatic approaches. The oligosaccharide fragments obtained were fractionated by gel permeation chromatography and analyzed for their molecular mass with MALDI-TOF MS. The obtained data show that this flax galactan is a complex RG-I polysaccharide with variable side chain structures. The backbone is composed of the common GalA-Rha repeats with a high degree of branching. These side chains are mainly composed of ß-1,4-linked Gal oligomers: (1) short branches of only one or two Gal residue(s); (2) long (linear) branches of up to 26 Gal residues; (3) mixed branches of between 3 and 12 Gal residues (possibly derived from longer linear side chains), that are resistant to galactanase cleavage; (4) side chains of at least 17 Gal residues, decorated with single Ara moieties. The linkage between RG backbone and galactan side chains was confirmed by the presence of fragments with (Rha-GalA)nHexm structure type. Neither chemical, nor enzymatic hydrolysis yielded oligomeric GalA residues, indicating that RG-I blocks are not interrupted by HGA regions. The polymer can be cleaved only partially by the rhamnogalacturonan hydrolase used, while the remaining part is resistant, probably due to peculiarities of side chain structure. Novel Rha-GalA oligomers were liberated by RG-hydrolase containing two or three Gal attached to Rha near the cleavage site. The native polymer is decorated by acetyl groups, with yet unknown distribution patterns. Treatment with purified and well-characterized galactanase does not change the hydrodynamic volume of flax galactan (despite considerable cleavage of Gal moieties), suggesting a complex ¿secondary¿ structure of the polymer.
Interactive forces between co-aggregating and non-co-aggregating oral bacterial pairs
Postollec, F. ; Norde, W. ; Vries, J. de; Busscher, H.J. ; Mei, H.C. van der - \ 2006
Journal of Dental Research 85 (2006)3. - ISSN 0022-0345 - p. 231 - 234.
microbial pairs - microscopy - adhesion - coaggregation - surfaces - cells - polysaccharides - spectroscopy - recognition - temperature
The temporo-spatial development of plaque is governed by adhesive interactions between different co-aggregating bacterial strains and species. Physico-chemically, these interactions are due to attractive Lifshitz-Van der Waals and acid-base forces, and occur despite electrostatic repulsion and with a critical influence of temperature. The forces between co-aggregating and non-co-aggregating pairs have never been measured, however. The aim here, thus, is to investigate, by atomic force microscopy, whether there is a difference in interactive forces between co-aggregating and non-co-aggregating bacterial pairs at 10 degrees C, 22 degrees C, and 40 degrees C. Actinomyces naeslundii 147 was immobilized on poly-L-lysine-coated tipless AFM cantilevers, while streptococci were immobilized on poly-L-lysine-coated glass surfaces. Upon approach, a repulsive force was measured, regardless of whether a co-aggregating or non-co-aggregating pair was involved. However, upon retraction, the co-aggregating pair exhibited larger adhesive forces and energies than did the non-co-aggregating pair. Adhesive interactions between the co-aggregating pair were smallest at 40 degrees C.
A new technique for extracting the red edge position from hyperspectral data : the linear extrapolation method
Cho, M.A. ; Skidmore, A.K. - \ 2006
Remote Sensing of Environment 101 (2006)2. - ISSN 0034-4257 - p. 181 - 193.
plant leaf reflectance - chlorophyll concentration - vegetation indexes - nitrogen status - canopy scales - area index - leaves - spectroscopy - variability - stress
There is increasing interest in using hyperspectral data for quantitative characterization of vegetation in spatial and temporal scopes. Many spectral indices are being developed to improve vegetation sensitivity by minimizing the background influence. The chlorophyll absorption continuum index (CACI) is such a measure to calculate the spectral continuum on which the analyses are based on the area of the troughs spanned by the spectral continuum. However, different values of CACI were obtained in this method because different positions of continuums were determined by different users. Furthermore, the sensitivity of CACI to agronomic parameters such as green leaf chlorophyll density (GLCD) has been reduced because the fixed positions of continuums are determined when the red edge shifted with the change in GLCD. A modified chlorophyll absorption continuum index (MCACI) is presented in this article. The red edge inflection point (REIP) replaces the maximum reflectance point (MRP) in near-infrared (NIR) shoulder on the CACI continuum. This MCACI has been proved to increase the sensitivity and predictive power of GLCD.
A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants
Hilz, H. ; Jong, L.E. de; Kabel, M.A. ; Schols, H.A. ; Voragen, A.G.J. - \ 2006
Journal of Chromatography. A, Including electrophoresis and other separation methods 1133 (2006)1-2. - ISSN 0021-9673 - p. 275 - 286.
plant-cell walls - cultured sycamore cells - cellulose interactions - covalent linkage - fruit xyloglucan - side-chain - oligosaccharides - polysaccharides - hemicellulose - spectroscopy
Different separation (HPAEC, RP¿HPLC, CE) and identification (MALDI¿TOF¿MS, ESI¿MSn) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC¿MALDI¿TOF¿MS) or online (CE¿ESI¿MSn, RP¿HPLC¿ESI¿MSn) connection provided complementary information on xyloglucan structure. Online CE¿MS and RP¿HPLC¿MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains.
The folding energy landscape of apoflavodoxin is rugged. hydrogen exchange reveals non-productive misfolded intermediates.
Bollen, Y.J.M. ; Kamphuis, M.B. ; Mierlo, C.P.M. van - \ 2006
Proceedings of the National Academy of Sciences of the United States of America 103 (2006)11. - ISSN 0027-8424 - p. 4095 - 4100.
azotobacter-vinelandii apoflavodoxin - protein - pathways - equilibrium - cooperativity - spectroscopy - sensitivity - topology - ensemble - dynamics
Many native proteins occasionally form partially unfolded forms (PUFs), which can be detected by hydrogen/deuterium exchange and NMR spectroscopy. Knowledge about these metastable states is required to better understand the onset of folding-related diseases. So far, not much is known about where PUFs reside within the energy landscape for protein folding. Here, four PUFs of the relatively large apoflavodoxin (179 aa) are identified. Remarkably, at least three of them are partially misfolded conformations. The misfolding involves side-chain contacts as well as the protein backbone. The rates at which the PUFs interconvert with native protein have been determined. Comparison of these rates with stopped-flow data positions the PUFs in apoflavodoxin's complex folding energy landscape. PUF1 and PUF2 are unfolding excursions that start from native apoflavodoxin but do not continue to the unfolded state. PUF3 and PUF4 could be similar excursions, but their rates of formation suggest that they are on a dead-end folding route that starts from unfolded apoflavodoxin and does not continue all of the way to native protein. All PUFs detected thus are off the protein's productive folding route
Structural characterization of tissue-specific galactan from flax fibers by 1H NMR and MALDI TOF mass spectrosmetry
Gur'janov, O.P. ; Gorshkova, T. ; Kabel, M.A. ; Schols, H.A. ; Dam, J.E.G. van - \ 2006
Russian Journal of Bioorganic Chemistry 32 (2006)6. - ISSN 1068-1620 - p. 558 - 567.
plant-cell walls - linum-usitatissimum l - rhamnogalacturonan-i - polysaccharides - oligosaccharides - identification - spectroscopy - transition - pectins - complex
A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by 1H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (- 4GalA1 - 2Rha1 -)n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 ß-(1 - 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.