bluuebunny/crossref_metadata_2025 huggingface dataset:
Further structural and functional properties of mini‒lipoxygenase, an active fragment of soybean lipoxygenase‐1
Authors: Maccarrone Mauro , Di Venere Almerinda , van Zadelhoff Guus , Mei Giampiero , Veldink Gerrit , Rosato Nicola , Finazzi-Agrò Alessandro
Published: March 2012

Lipoxygenases (Loxs) form a homologous family of non‐heme, non‐sulfur iron containing lipid‐peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase‐1 (Lox‐1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox‐1 is known to produce a 60 kDa fragment, termed “mini‐Lox”, which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi‐Agrò, Biochemistry40 (2001), 6819–6827). In this study, we have investigated the stability of mini‐Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini‐Lox is a rather unstable molecule, which undergoes a two‐step unfolding transition. Both chemical‐ and physical‐induced denaturation suggest that mini‐Lox is more hydrated than Lox‐1, an observation also confirmed by 1‐8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate‐induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini‐Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox‐1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini‐Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency.
Spherical polyelectrolyte brushes as carrier particles for proteins: An investigation of the structure of adsorbed and desorbed bovine serum albumin
Authors: Jackler G. , Wittemann A. , Ballauff M. , Czeslik C.
Published: March 2012

The change in the secondary and tertiary structure of bovine serum albumin (BSA) induced by the interaction with spherical polyelectrolyte brushes (SPB) has been investigated using fluorescence and circular dichroism (CD) spectroscopy. The SPB consist of poly(acrylic acid) chains grafted to a poly(styrene) core. The colloidal SPB represent a new substrate for protein immobilization because their protein binding capacity can be controlled by the ionic strength of the solution: SPB bind large amounts of BSA at low ionic strength (pH=6.1), but they are largely protein resistant at moderate salt concentrations of 500 mM. The conformation of BSA which was labeled with the environmentally sensitive dansyl fluorophore was studied before adsorption to the SPB, in the adsorbed state, and after desorption from the SPB. In the adsorbed state the obtained fluorescence spectrum is red‐shifted which indicates a hydration of the dansyl fluorophores due to a distortion of the tertiary structure of BSA. Fluorescence and CD spectroscopic analysis of BSA that was desorbed from the SPB shows that the adsorption‐induced conformational changes are largely reversible. Convex constraint analysis of the observed CD spectra of BSA yield α‐helix fractions of 68% and 57% before adsorption to and after desorption from the SPB, respectively. In a general view, the results of this study demonstrate that spherical polyelectrolyte brushes are suitable for a controlled immobilization and release of proteins without major conformational changes.
Physical characterisation of microporous and nanoporous polymer films by atomic force microscopy, scanning electron microscopy and high speed video microphotography
Authors: Barrow M. S. , Jones R. L. , Park J. O. , Srinivasarao M. , Williams P. R. , Wright C. J.
Published: March 2012

We report studies of ordered microporous and nanoporous polymer films formed by the evaporation of polymer solutions following exposure to a humid atmosphere. High speed microphotographic (HSMP) studies of the formation process showed that near the surface of the polymer solution, vapour condensation produced near mono‐disperse water droplets which form a close‐packed monolayer (or ‘breath figure’). Following the evaporation of the solvent, characterisation of the solid by Atomic Force Microscopy and Scanning Electron Microscopy revealed that the surface of the polymer film is characterised by extensive regions of hexagonally close‐packed microscopic pores, whose spatial arrangement replicates that of the initial droplet monolayer. Characterisation of sections of the film by Atomic Force Microscopy established that the surficial pores represent open sections of sub‐surficial spheroidal cavities formed by encapsulation of the water droplets within the polymer solution. An interesting feature of the results is the occurrence of nano‐scale pores at the film surface and at (and within) the walls of the sub‐surficial microscopic pores. This is the first physical evidence report of such features in porous polymer films produced by a process involving breath‐figures. Their dimensions suggest that more detailed structural investigations will require alternative techniques to conventional, optical methods.
FT‐IR spectroscopy for the detection of liver damage
Authors: Crupi V. , Majolino D. , Migliardo P. , Mondello M. R. , Pergolizzi S. , Venuti V.
Published: March 2012

A spectroscopic analysis of hepatotoxic effects on rat liver, caused by a single administration of carbon tetrachloride (CCl4), has been performed by Fourier Transform Infrared (FT‐IR) absorption technique in a wide range of frequency 3000–400 cm–1. The spectral investigation has permitted us to mark the dynamical changes of rat liver samples vs. the time course of the injuries. In particular as lower frequency is concerned, the comparison among the control sample and the injured ones has revealed a clear change in the typical lipids stretching region 1800–1000 cm–1. Furthermore, in the CH stretching region 3000–2400 cm–1, we showed a clear change in the band shape. The observed variations, respect to the normal liver case, have been interpreted on the basis of previous structural and biochemical analysis and in the framework of a recent FT‐IR analysis of the O–H stretching region 3800–3000 cm–1.
RAG1 oligomerization states and secondary structural properties: an initial characterization of V(D)J recombinase complex formation
Authors: Godderz LeAnn J. , Rodgers Karla K.
Published: March 2012

The recombination activating gene products (RAG1 and 2) catalyze the initial DNA cleavage steps during V(D)J recombination for the diversification of the immune repertoire. As the fundamental properties of the RAG proteins remain largely unknown, our objective is to investigate the self–association and conformational properties of RAG1. To analyze RAG1 association and dissociation, a time course of multi–angle laser light scattering measurements (MALL SEC) was performed on samples at different oligomerization states over a wide range of ionic strengths. The molecular masses of the predominant RAG1 species corresponded to dimer, tetramer, and a previously uncharacterized octamer state. Furthermore, the fraction of RAG1 in the tetrameric and octameric states increased significantly over time at lower ionic strengths, indicating that these oligomeric forms may factor into the physiological function of RAG1. Circular dichroism (CD) analysis of RAG1 showed a significant dependence in secondary structure on ionic strength with changes in α‐helical content over time that may correspond to the changes in oligomerization states shown by MALLS SEC. Together, the MALLS SEC and CD analyses of RAG1 self‐association properties and secondary structure give further insight into formation of the protein complex responsible for catalyzing V(D)J recombination.
On dissipation of quartz crystal microbalance as a mechanical spectroscopy tool
Authors: Voinova M. V. , Jonson M. , Kasemo B.
Published: March 2012

We report on theoretical analysis of dissipative effects in quartz crystal resonator applications to the dynamics of complex biological fluids and soft polymer films. As a mechanical spectroscopy tool, the quartz resonator can probe the storage and loss moduli μ(ω) of a thin material sample in small amplitude oscillations where polymers exhibit linear viscoelasticity. We show how viscosity (internal friction) and slippage (interfacial friction) of the sample affect the acoustical characteristics of the quartz resonator. With respect to biosensor’s application, we present rigorous expressions for the resonant frequency and damping of the quartz crystal which allow to quantify friction effects and even distinguish between them in resonator measurements performing on various frequencies. Possible application of the results in electronic nose and electronic tongue sensors is discussed.
Vibrational spectroscopy and computer modeling of proteins: solving structure of α1‐acid glycoprotein
Authors: Kopecký Vladimír , Ettrich Rüdiger , Hofbauerová Kateřina , Baumruk Vladimír
Published: March 2012

This work introduces a new approach connecting vibrational spectroscopy with homology and energetic molecular modeling of proteins. Combination of both methods can compensate their disadvantages and result in realistic three‐dimensional protein models. The approach is most powerful for membrane proteins or glycoproteins with high carbohydrate content where X‐ray or NMR analysis is not always successful. Nevertheless, it can also serve as a tool of preliminary analysis of any protein with unknown structure. Power of the approach is demonstrated on human α1‐acid glycoprotein. Its predicted structure published in [V. Kopecký Jr. et al., Biochem. Biophys. Res. Commun. 300 (2003), 41–46] is discussed in detail with respect to the approach and its general employment.
Structural dissection of alkaline‐denatured pepsin
Authors: Kamatari Yuji O. , Dobson Christopher M. , Konno Takashi
Published: March 2012

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IP state) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IP state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IP state of pepsin. CD spectra indicate in addition that significant non‒native α‐helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IP state is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.
Analysis of non‐protein amino acids as specific markers of low density lipoprotein apolipoprotein B‐100 oxidation in human atherosclerotic lesions: the use of N(O)‐ethoxycarbonyl trifluoroethyl ester derivatives and GC‐MS 1
Authors: Pietzsch Jens , Bergmann Ralf , Kopprasch Steffi
Published: March 2012

Oxidative modification of proteins can interfere with critical cellular functions, and is widely regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to atherosclerosis and cancer. In this line, a new GC‒MS methodology using N(O)‒ethoxycarbonyl trifluoroethyl amino acid esters (ECEE‒F3) for rapid and sensitive determination of 3‒chlorotyrosine, 5‒hydroxy‒2‒aminovaleric acid (HAVA), and 6‒hydroxy‒2‒aminocaproic acid (HACA) in proteins has been developed. 3‒Chlorotyrosine is a highly specific marker of myeloperoxidase catalyzed protein oxidation, whereas γ‒glutamyl semialdehyde (γGSA) and α‒aminoadipyl semialdehyde (αASA), which by reduction form HAVA and HACA, respectively, are specifically formed by metal catalyzed oxidation processes. ECEE‒F3 derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. The key steps of the methodology employed are (i) enzymatic hydrolysis of target proteins to prevent decomposition of oxidation products during hydrolysis and (ii) an uniquely rapid derivatization of amino acids completing sample preparation for GC within a few minutes in aqueous solution at room temperature. The use of these stable products of protein amino acid side chain oxidation as potential markers for assessing oxidative damage in LDL apoB‒100 recovered from human aortic vascular lesions is demonstrated. These observations provide quantitative chemical evidence for metal catalyzed oxidative processes in the human artery wall.
The association of dimethylsulfoxide and model membranes studied by pulse‐field gradient NMR
Authors: Kennedy Anthony , Long Carla J. , Hmel Peter J. , Hicks Rickey , Reid Thomas J.
Published: March 2012

The use of dimethylsulfoxide (DMSO) as a cryoprotectant to reduce cellular injury during freezing is well known, however the intermolecular interactions between this amphiphilic molecule and biological membranes that form the basis of this protection are unknown. DMSO–dipalmitoylphosphatidylcholine (DPPC) vesicle interactions were investigated in pulsed‐field gradient NMR (PFGNMR) experiments and spectra analysis allowed for the determination of self‐diffusion coefficients for each species present. The mole fraction of DMSO associated with the DPPC vesicles was then calculated from the diffusion coefficients: the mole fraction increased from 14% to 42% as the membrane was heated from below to above the main phase transition temperature.
Human breast tissue characterisation with small‐angle X‐ray scattering
Authors: Fernández M. , Keyriläinen J. , Karjalainen-Lindsberg M.-L. , Leidenius M. , von Smitten K. , Fiedler S. , Suortti P.
Published: March 2012

Small‐angle X‐ray scattering (SAXS) patterns from breast tissue samples are compared with their histology. Formalin fixed human breast tissue specimens containing ductal and lobular carcinoma were studied. Histo‐pathological information is compared with the scattering data, and there is a clear spatial correlation. Supra‐molecular organisation of collagen fibrils is modelled and the model is used to create scattering maps. The model parameters include the axial periodicity (d‐spacing), radius and packing of the fibrils, and these are derived from comparison with the experimental scattering patterns. The d‐spacing is to 0.5% larger in malignant zones of the tumours than in the healthy zones. There are also characteristic differences in the fibril diameter and packing.
XPS study of the adsorption mechanisms of DNA onto polypyrrole particles
Authors: Saoudi Bachir , Jammul Noraldeen , Chehimi Mohamed M. , Jaubert Anne-Sophie , Arkam Chakib , Delamar Michel
Published: March 2012

DNA adsorption onto polypyrrole (PPy) powder particles has been monitored, ex situ, by X‐ray photoelectron spectroscopy (XPS) technique. DNA adsorption isotherms were determined by the quantitative analysis of the XPS spectra, and by plotting the X/N atomic ratios (X = C, O, Cl, P and Na) versus DNA equilibrium concentration. All XPS isotherms are of high affinity type, showing high adsorption amounts at low DNA concentrations in the suspension. Moreover, inspection of the C1s peak structure of the PPy–DNA complex revealed that it gradually gets wider and less tailing as DNA adsorbs, clearly showing the DNA contribution to the peak enlargement. In addition, the changes observed in the Cl2p structure bring a strong supporting evidence of anion‐exchange mechanism that takes place at initial stages of the interaction. Actually, the polypyrrole backbone loses part of its residual chlorides as the first DNA fragments adsorb and neutralize the PPy positive charges at the interface. Moreover, at relatively high amounts of adsorbed DNA, the PPy surface becomes screened necessitating thus that sodium cations co‐adsorb in order to compensate for the excess of DNA negative charges. As a consequence of such screening of the PPy surface, DNA adsorption results in a positive spectral shift of all peaks of approximately 2.0 eV, a value that leads to the conclusion that DNA partially covers the PPy.
Glucose biosensor based on entrapment of glucose oxidase and myoglobin in silica gel by the sol‐gel method
Authors: Zaitoun Mohammed A.
Published: March 2012

A spectrophotometric method is presented to determine glucose employing the sol‐gel technique. Myoglobin (Mb) and glucose oxidase are encapsulated in a transparent and porous silica glass. The produced gel (xerogel) is then immersed in water where increments of glucose are added to the solution with stirring; glucose diffuses into the sol‐gel glass pores and a series of reactions take place. Glucose is first oxidized by glucose oxidase and oxygen to gluconate and hydrogen peroxide is generated. The liberated hydrogen peroxide oxidizes the Mb heme (Fe2+ into Fe3+). The higher is the glucose concentration added, the more is the H2O2 generated, and the more is the Mb oxidation (Fe2+ to Fe3+) and as a result the higher is the absorbance at 400 nm (negative peak, lower absorbance value). All measurements are performed at this wavelength (400 nm), the negative peak obtained by subtracting the absorption spectra of Mb before and after oxidation. Measuring the slope of the absorbance decay versus time at 400 nm monitors increments of added glucose. Each glucose concentration has an accompanying unique decay curve with a unique slope. The higher is the glucose concentration; the steeper is the decay curve (higher slope value). The calibration curve was linear up to 40 mM.
Intramuscular haemodynamics using near infra‐red spectroscopy as a research strategy to understand chronic muscle pain pathophysiology
Authors: Kuboki Takuo , Maekawa Kenji , Clark Glenn
Published: March 2012

Recently, localized intramuscular haemodynamic disturbance has been recognized as one of the cardinal features of a chronically painful muscle. To test this hypothesis, we have carried out several studies that have assessed intramuscular haemodynamics to understand more about haemodynamic alteration under conditions of an experimentally induced vasoreactive challenges in healthy controls and in patients with chronic muscle pain using near infra?red (NIR) spectroscopy. In addition, we have validated the NIR based haemodynamic signals by comparing against a water signal intensity change taken from a transverse T2?weighted magnetic resonance image (MRI) of the muscles. As a result, we discovered that muscle pain patients have a diminished ability to vasodilate in painful muscle. We also provided powerful evidences that NIR data (total haemoglobin level), transcutaneously taken from the muscles in humans could be an analogue of intramuscular haemodynamics.
Dynamic lipidomic insights into phosphatidylcholine synthesis from organelle to organism
Authors: Hunt Alan N. , Postle Anthony D.
Published: March 2012

Recent technical improvement and technological innovation in small molecule mass spectrometry have provided powerful tools for the intensive metabolomic biochemical investigations that will necessarily characterise the ”post‐genomic“ era of biomedical research. For membrane phospholipids, use of tandem electrospray ionisation mass spectrometry (ESI‐MS/MS) exploiting precursor scanning of class‐specific diagnostic fragments, can provide detailed quantitative profiles at the level of individual molecular species for many hundreds of unique lipids. Such ”snapshot“ measurements provide little information concerning metabolic flux. However, recent use of metabolic labelling with stable isotope derivatives of phospholipid headgroups combined with precursor scans of unlabelled and labelled fragments have yielded considerable insight into phosphatidylcholine metabolism in vivo. Here, we briefly review some of the recent work on pathways of phosphatidylcholine metabolism ranging from studies at subcellular organelle level through to whole organism. The sensitivity, specificity and suitability of this powerful methodological approach to numerous questions of phospholipid metabolism place ESI‐MS/MS at the very heart of dynamic lipidomics in the foreseeable future.
Monitoring EDTA and endogenous metabolite biomarkers from serum with mass spectrometry
Authors: Lowe Rachel , Go Eden P. , Tong Grace C. , Voelcker Nicolas H. , Siuzdak Gary
Published: March 2012

We describe a quantitative method for the determination of ethylenediaminetetraacetic acid (EDTA) in human serum by gas chromatography mass spectrometry (GC‐MS), liquid chromatography electrospray ionisation tandem mass spectrometry (LC‐ESI‐MS/MS), and desorption ionisation on silicon mass spectrometry (DIOS‐MS). In the initial stages of the analysis, endogenous metabolites (1‐palmitoyl‐sn‐glycero‐3‐phosphocholine and 1‐stearoyl‐sn‐glycero‐3‐phosphocholine) were readily observed in LC‐ESI‐MS and DIOS‐MS however, direct analysis of the EDTA free acid had limited sensitivity. In order to improve EDTA detection we employed a straightforward esterification derivatization. The most successful derivatization procedure converted EDTA to its methyl ester and, since 13C isotopes of these reagents are readily available, internal standards could be easily generated for quantitative analysis. This approach provided a limit of detection of 0.5 and 0.1 μM for GC‐MS and LC‐ESI‐MS, and offers a viable method for the EDTA detection.
On the mechanism of a new dihalocyclopropane‐dihalomethyl vinyl rearrangement
Authors: Jankowski C.K. , Bou Laouz A. , Diaz-Torres E. , Lesage Denis , Bélanger J.M.R. , Paré J.R.J.
Published: March 2012

A tentative mechanism for a new dihalocyclopropane‐dihalomethyl vinyl rearrangement is discussed following some isotopic labelling and substituted cyclohexene studies on fused cyclohexene‐cyclopropane (norcarane) compounds.
Effect of temperature on liposome structures studied using EPR spectroscopy
Authors: Sulkowski W.W. , Pentak D. , Korus W. , Sulkowska A.
Published: March 2012

The effect of temperature on liposome structures has been investigated by means of electron paramagnetic resonance spectroscopy with the use of the spin labelling technique. The EPR spectra were recorded on a Bruker EMX spectrometer at the X band in the temperature range 300–340 K. Liposomes were prepared from L?a?phosphatidylcholine dipalmitoyl (1,2?dihexadecanoyl?sn?glycerol?3?phosphocholine) 99% (DPPC), DL?a?phosphatidylcholine dimyristoyl (1,2?ditetradecanoyl?rac?glycerol?3?phosphocholine) 99% (DMPC) and cholesterol (5?cholesten?3ß?ol) 99+% to constitute the membrane. The spin marker, 2?(3carboxypropyl)?4,4?dimethyl?2?tridecyl?3?oxazolidinyloxyl free radical (5?DOXYL), placed in a liposome membrane, allows for observation of structural changes in liposomes with temperature increase. The changes of rotational correlation time and order parameter values with increasing temperature result from the motion rise of the spin probe. The intensity of the EPR signal of 5?DOXYL gives us information about membrane fluidity.
Concentration dependent different action of progesterone on the order, dynamics and hydration states of the head group of dipalmitoyl‐phosphatidylcholine membrane
Authors: Korkmaz Filiz , Kırbıyık Halil , Severcan Feride
Published: March 2012

Interactions of progesterone with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of progesterone concentration at selected temperatures monitoring both the gel and liquid crystalline phase, by using Fourier Transform Infrared spectroscopy (FTIR). It has been show that the effect of progesterone on membrane dynamics is dependent on progesterone concentration. At 1 mol%, which is close to physiological level, progesterone behaves differently. At this concentration the decrease in dynamics is more noticeable. Additionally a dramatic decrease in the strength of hydrogen bonding in the interfacial region of the bilayer is also observed. When concentration increases up to 12 mol% opposite behaviour is observed at all interactions. Above 12 mol%, progesterone–DPPC interactions shows almost linear plot.
13C nuclear magnetic resonance spectroscopy to check 1,3‐random, 2‐random pattern of fatty acid distribution in olive oil triacylglycerols
Authors: Vlahov Giovanna
Published: March 2012

The fatty acid distribution between the 1,3‐ and 2‐positions of triglycerides was determined in the olive oil set sampled in the Marche region, by using the 13C NMR method which applies the DEPT pulse sequence. The results confirmed that the oleic and linoleic acids were not randomly distributed at the 2‐position of triacylglycerols but were lower for oleic acid and higher for linoleic acid compared to the theoretical values expected for a random distribution. Moreover, the unsaturated acids deviated from the 2‐random distribution at different extents according to the acid concentration of triglyceride.

The 13C NMR results, they were obtained for three olive oil samples characterized by high, medium and low oleic acid at 2‐position (Ofound) corresponding to a low, medium, high deviation rate from the 2‐random pattern, were compared to those calculated by the computer program based on the 1,3‐random‐2‐random distribution of fatty acids in the triglycerides.

The molar concentrations of the triglyceride species LLL and OLL were calculated from the molar concentrations of oleic and linoleic acids at the 2‐ and 1,3‐positions obtained by using 13C NMR and the computer method. The LLL and OLL triglycerides determined by the computer method were found to be markedly higher in the “high Ofound” oil as compared to NMR. No differences were detected between the two methods for both triglyceride species in the “low Ofound” oil.
A dialysis accessory for attenuated total reflection infrared spectroscopy
Authors: Krasteva Maria , Kumar Saroj , Barth Andreas
Published: March 2012

A dialysis accessory for attenuated total reflection (ATR) infrared spectroscopy is described together with an evaluation based on known systems with well‐studied infrared spectra, such as chemical oxidation and reduction of cytochrome c and substrate binding to the Ca2+‐ATPase. Changes in the infrared spectra of the two proteins are successfully monitored with the dialysis accessory. The accessory was developed in our laboratory for the diamond 9‐reflections SensIR ATR unit. It can be used to study absorbance changes of macromolecules which are induced by low molecular weight compounds, for example the binding of substrates, inhibitors or ions to macromolecules as well as effects of pH, ionic strength or denaturants on the structure of macromolecules. The dialysis accessory confines the macromolecule of interest to a sample compartment created between the ATR crystal and the dialysis membrane. On the other side of the dialysis membrane, a reservoir for the sample medium is created. In this way the low molecular weight compound of interest can exchange freely between the reservoir and the sample compartment via the dialysis membrane. This provides a flexible way to change sample conditions for the macromolecule of interest, allowing for example initiation of ligand binding.
Electron paramagnetic resonance and mass spectrometry: Useful tools to detect ultraviolet light induced skin lesions on a molecular basis – A short review
Authors: Hochkirch Ulrike , Herrmann Werner , Stößer Reinhard , Borchert Hans-Hubert , Linscheid Michael W.
Published: March 2012

Ultraviolet radiation is considered responsible for sunburning, premature skin aging, and cancerogenesis through the production of free radical species. Therefore, the favoured possibility for direct detection of unpaired electrons – electron paramagnetic resonance spectroscopy – is predestinated for detection and structural and dynamic analysis of this kind of molecules. However, many of UV induced radicals in skin have a short lifetime at ambient conditions and possibilities for stabilisation or transformation into definite para‐ or diamagnetic products have to be found. On the other hand, diamagnetic products, potentially also originated by reporter molecules, which are not detectable by EPR, are target molecules for mass spectrometric analysis. In this review, potentials and limitations of both spectroscopic methods are reviewed, and the effect of ultraviolet radiation on human skin is discussed in particular. Suitable combinations of both techniques result in detailed information about photoproducts and processes taking place within skin during and after irradiation. The literature is viewed from a recent perspective; historical aspects were not in the scope of this paper.
Ultrasensitive determination of amoxicillin using chemiluminescence with flow injection analysis
Authors: Xie Xiaofeng , Song Zhenghua
Published: March 2012

Results presented here reveal that amoxicillin can greatly enhance the chemiluminescence intensity generated from the reaction between luminol and hydrogen peroxide. The increment chemiluminescence signal was linearly dependent on amoxicillin concentration in the range from 10 pg·ml−1 to 2 ng·ml−1 (r2=0.9978) offering a detection limit as low as 3.5 pg·ml−1 (3σ). At a flow rate of 2.0 ml·min−1, one analysis cycle, including sampling and washing, can be accomplished in 20 s with a relative standard deviation of less than 5%. The sensitive flow injection method was applied successfully to determine of amoxicillin in pharmaceutical preparations, human urine and serum without any pretreatment procedure, with recovery from 90.0% to 110.0% and relative standard deviations of less than 5.0%.
Application of a low‐angle light scattering technique to cell volume and cell signaling studies on Ehrlich ascite tumor cells
Authors: Zinchenko Valeriy P. , Lee Vyacheslav V. , Berezhnov Alexey V. , Mindukshev Igor V. , Jenkins Richard O. , Goncharov Nikolay V.
Published: March 2012

A method for studying cells based on low‐angle light scattering was applied to cell volume and cell signaling studies on Ehrlich ascite tumor cells (EATC). Changes in the volume of EATC were measured in hypotonic medium, as well as after activation with exogenous ATP, ionomycin and thimerosal. Increase of [Ca2+]i under ATP and ionomycin action induced reversible changes of cell volume: fast shrinking was followed by swelling. Thimerosal caused a reversible change in EATC volume with high amplitude; endoplasmic reticulum played the key role in this response. Having obtained kinetic parameters of changes in cell volume under activation of the cells, quantitative measurements of K+, Na+ and anion flows responsible for this process can then be obtained. In spite of some fundamental differences in the behavior of cells of different dimensions there are many similarities, and there is a good theoretical background for dealing with both small and large cells.
Full validation of an electrothermal atomic absorption assay for zinc in hepatic tissue using a fast sample preparation procedure
Authors: Fröehlich Pedro Eduardo , Pinto Raquel Borges , Wortmann André Castagna , Magalhães Richard Borba , Schneider Ana Cláudia Reis , Ferreira Jéferson José , da Silveira Themis Reverbel
Published: March 2012

The objective of this work was to develop and fully validate an analytical assay to quantify zinc in hepatic tissue. The procedure should be as simple and fast as possible in order to avoid sample contamination. The amount of sample used should also reflect the sample size usually obtained in clinical biopsies, which are about 3–4 mg at most. The validation protocol is in accordance to international guidelines, such as ICH and FDA. The parameters evaluated were precision, accuracy, range, limit of detection and limit of quantification. The method was evaluated in the 2.0–32.0 parts per billion (μg/l) range. Under the described conditions intra and inter day precision of the three levels of quality controls were lower than 9.06 and 5.27, respectively, expressed as relative standard deviation (RSD). The accuracy ranged from 86.35 to 114.71%. Limit of detection and limit of quantification were 0.60 and 2.0 μg/l, respectively. Fresh bovine liver samples were used in order to evaluate the clinical procedure used to collect biopsies. According to the results and experimental protocol, the method is fully validated and ready to use in clinical trials involving zinc quantitation using hepatic samples as small as 2.00 mg of dry tissue.
Free radical reactions in atherosclerosis; An EPR spectrometry study
Authors: Taiwo F. A. , Powers H. J. , Nakano E. , Griffiths H. R. , Nugent D. F.
Published: March 2012

The copper catalysed oxidation of homocysteine has been studied by electron paramagnetic resonance (EPR) spectroscopy and spin trapping techniques to determine the nature of free radical species formed under varying experimental conditions. Three radicals; thiyl, alkyl and hydroxyl were detected with hydroxyl being predominant. A reaction mechanism is proposed involving Fenton chemistry. Inclusion of catalase to test for intermediate generation of hydrogen peroxide showed a marked reduction in amount of hydroxyl radical generated. In contrast, the addition of superoxide dismutase showed no significant effect on the level of hydroxyl radical formed. Enhanced radical formation was observed at higher levels of oxygen, an effect which has consequences for differential oxygen levels in arterial and venous systems. Implications are drawn for a higher incidence of atherosclerotic plaque formation in arteries versus veins.
Stabilization of N‐, N,N‐, N,N′‐methylated and unsubstituted simple amidine salts by multifurcated hydrogen bonds
Authors: Spychała Jarosław
Published: March 2012

In the light of the usefulness of amidines in medicinal chemistry, this paper considers the effects on biological properties and chemical reactivities of organic molecules affected by intramolecular interactions. The study of chemical shifts has been an important source of information on the electronic structure of amidine salts and their ability to form non‐covalent bonds with nucleic acids. The NMR and IR results demonstrate that hydrogen bonds are a force for promoting chemical reactions. The thymine O2 carbonyl oxygen in a close proximity to the amidinium cation does interact with the appropriately spaced amidinium NH donor moieties. The 1H‐15N 2D NMR (GHSQC and GHMBC) spectra with natural isotopic abundance of 15N fully confirm the intramolecular character of the bonds. A rule able to estimate the relative strength of the new multifurcated hydrogen bonds is given. The appearance of the ΔδNH chemical shift differences near zero is due to the strong intramolecular interactions. The strength of the H‐bond donation by acetamidines is reflected in the N–H dissociation/recombination process (positive charge shift has been invoked to explain other effects on benzamidines). The temperature dependence of chemical shift for the amidine NH protons in dimethyl sulfoxide solutions is herein discussed.
Understanding arsenic metabolism through spectroscopic determination of arsenic in human urine
Authors: Brima Eid I. , Jenkins Richard O. , Haris Parvez I.
Published: March 2012

In this review we discuss a range of spectroscopic techniques that are currently used for analysis of arsenic in human urine for understanding arsenic metabolism and toxicity, especially in relation to genetics/ethnicity, ingestion studies and exposure to arsenic through drinking water and diet. Spectroscopic techniques used for analysis of arsenic in human urine include inductively coupled plasma mass spectrometry (ICP‐MS), hydride generation atomic absorption spectrometry (HG‐AAS), hydride generation atomic fluorescence spectrometry (HG‐AFS), graphite furnace atomic absorption spectrometry (GF‐AAS) and liquid chromatography mass spectrometry (LC‐MS). Advantages and disadvantages of each of these techniques are highlighted in relation to analysis of total arsenic and speciation of arsenic compounds in human urine which would be of particular benefit to those new to the field of arsenic speciation of urine samples. The review summarises our current understanding of arsenic metabolism through the analysis of urine samples and provides a list of arsenic species that have been identified in urine samples. This is done by focusing on recent published reports on arsenic metabolism and toxicity in relation to most investigated issues, i.e. genetics/ethnicity, ingestion and exposure.
Single scan TROSY and E.COSY suite of experiments for the measurement of residual dipolar couplings in proteins
Authors: Eliseo Tommaso , Gallo Mariana , Melis Riccardo , Paci Maurizio , Bazzo Renzo , Cicero Daniel O.
Published: March 2012

Simple modifications of the gradient enhanced version of the TROSY experiment allow to obtain four different spectra containing each one of the components of the H–N doublet in 15N labelled proteins. By measuring the difference in peak position among these four spectra, residual dipolar coupling values can be obtained for medium sized proteins. This experiment, which exploits the use of pulse field gradients to select the 15N coherence pathway, produces excellent results in terms of water suppression. Moreover, tuning of a single delay in the sequence reduces notably the presence of artifacts. The performance of this suite of experiments, that we called TEC (Trosy–E.Cosy) experiment, is tested against a J‐modulation method, inherently more accurate but more time consuming, for the accuracy in the observed values of residual dipolar couplings. For larger proteins, the use of this strategy allows to select the most favourable combination of peaks giving the sharpest signals.
Structural changes of protein antigens due to adsorption onto and release from aluminium hydroxide using FTIR–ATR
Authors: Zheng Yiwu , Lai Xuxin , Ipsen Henrik , Larsen Jørgen Nedergaard , Løwenstein Henning , Søndergaard Ib , Jacobsen Susanne
Published: March 2012

Structural integrity of antigens upon adsorption and release is not only important for investigating vaccine immunogenicity, but also for the epitope specificity of the resulting immune response and hence therapeutic efficacy. Moreover, the structural information is also important for understanding the mechanism of how adjuvants can enhance the immune response. However, little is known about an antigen′s structure when it is adsorbed on and subsequently released from aluminium adjuvants. In this study, the structures of two protein antigens, bovine serum albumin and β‐lactoglobulin, were investigated using Fourier transform infrared–attenuated total reflection (FTIR–ATR) spectroscopy. The secondary structures of both model antigens change when adsorbed to aluminium hydroxide. The structural perturbation depends on the amount of adsorbed protein. Maximal adsorption gives a more native‐like structure. This may indicate that protein is adsorbed in different manners depending on the concentration. The adsorbed antigens are released using phosphate buffer pH 7.4 (PB). The recovery is approximate 80% after 40 min in the presence of PB. The recovery curves of both proteins also indicate two different adsorption modes. FTIR–ATR and circular dichroism (CD) spectroscopy yield similar results suggesting that the adsorbed antigens refold to their native‐like state after release.
Silver(I) complexes of imidazolidine‐2‐thione and triphenylphosphines: Solid‐state, solution NMR and antimicrobial activity studies
Authors: Al-Zamil Norah O. , Al-Sadhan Khulood A. , Isab Anvarhusein A. , Wazeer Mohamed I.M. , Al-Arfaj Abdul Rehman A.
Published: March 2012

Mixed ligand complexes of Ag(I) with triphenylphosphine (PPh3), triphenylphosphine sulfide (SPPh3), triphenylphosphine selenide (SePPh3) and Imidazolidine‐2‐thione (Imt) have been prepared. The solution as well as solid state NMR studies have been carried out to characterize these complexes. Both solid and solution NMR show the coordination via thione group on one side and (S/Se) or PPh3 on the other side. A higher antimicrobial activity is shown by [ImtAgPPh3]Cl complex against gram negative Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) compared to the other two complexes i.e. [ImtAgSPPh3]Cl and [ImtAgSePPh3]Cl.
Vibrational analysis of deoxy‐andrographolide using MM/QM methods
Authors: Singh P. K. , Hasan Tanveer , Prasad Onkar , Sinha Leena , Jain Sudha , Raj Kanwal , Sundaraganesan N. , Misra Neeraj
Published: March 2012

Bioactive natural products have an enormous economic importance as speciality chemicals. Extracts of the shrub Andrographis paniculata are widely used as herbal medicines in many countries. Diterpenoid lactones isolated from these extracts have been the subject of extensive investigation, and one of its constituent diterpenoid is deoxy‐andrographolide. A complete normal coordinate analysis was performed for deo‐andrographolide in terms of the calculation by using Classical mechanics and Quantum mechanical methods.The study of vibrational spectra helps in understanding the dynamical behaviour and hence a lot of information can be derived about the conformation. The geometrical parameters and the vibrational spectra have been discussed in detail.
Spectroscopic studies on the interaction of hypocrellin A with myoglobin
Authors: Zhou J. H. , Wu X. H. , Yang C. , Gu X. T. , Zhou L. , Song K. X. , Feng Y. Y. , Shen J.
Published: March 2012

Experimental results of UV‐visible absorption spectroscopy and fluorescence spectroscopy indicate that hypocrellin A, which has been studied in photodynamic therapy, can interact with the surface of myoglobin through hydrophobic forces, and form a complex. Based on the Stern–Volmer equation, the quenching constants of the process can be calculated to be 4.81×1012 L mol−1 s−1 (t=25°C) and 4.54×1012 L mol−1 s−1 (t=42°C) respectively, and the binding constant is 5.53×104 M−1 (t=25°C), while the binding sites is 0.94 (t=25°C). In addition, Electron paramagnetic resonance and fluorescence spectroscopic analysis suggests that that the quenching mechanism of the interaction process occurs through the electron transfer between hypocrellin A and myoglobin.
Polarographic and spectroscopic studies of the effects of fluoroacetate/fluorocitrate on cells and mitochondria
Authors: Zinchenko Valeriy P. , Goncharov Nikolay V. , Teplova Vera V. , Kasymov Vitaliy A. , Petrova Olga I. , Berezhnov Alexey V. , Senchenkov Evgeniy V. , Mindukshev Igor V. , Jenkins Richard O. , Radilov Andrey S.
Published: March 2012

Experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells (EATC) and cardiomyocytes, exposed to fluoroacetate (FA) or fluorocitrate (FC) in vitro. The effects of FA developed at much higher concentrations in comparison with FC and was dependent upon respiratory substrates: with pyruvate, FA induced a slow oxidation of pyridine nucleotides (NAD(P)H) and inhibition of respiration. NAD(P)H oxidation was prevented by incubation of mitochondria with cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore. Studies of the NAD(P)H level and calcium response generated in EATC under activation with ATP via the metabotropic P2Y receptor, revealed a loss of NAD(P)H from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H on exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes, and cause the hypersensitivity of platelets reported on earlier. Highlighting the reciprocal relationship between intracellular NAD(P)H and calcium balance, we discuss metabolic pathway modulation in the context of development of an effective therapy for FA poisoning.
Hepatic iron quantification by atomic absorption spectrophotometry: Full validation of an analytical method using a fast sample preparation
Authors: Wortmann A. C. , Froehlich P. E. , Pinto R. B. , Magalhães R. B. , Álvares-da-Silva M. R. , Ferreira J. J. , Silveira T. R.
Published: March 2012

Atomic absorption spectrophotometry is considered the method of choice for hepatic iron quantification. The objective of the present study was to perform full validation assays of hepatic iron quantification by atomic absorption spectrophotometry, using a fast sample preparation procedure, following the guidelines from the International Conference on Harmonization. The following parameters were evaluated: specificity, linearity/range, precision, accuracy, limit of detection and limit of quantification. A good linear correlation was found (0.9948) in the concentration range evaluated (20‐ 120 ppb). The relative standard deviations were below 15% for accuracy, and below 10% for both day‐to‐day reproducibility and within‐days precision, and the repeatability of injections was 0.65%. Limit of detection was 2 ppb, and limit of quantification was 6 ppb. Fresh bovine liver tissue was used to evaluate the procedure of collecting samples by liver biopsies. These findings indicate that hepatic iron quantification by atomic absorption spectrophotometry can be reliably performed at the established conditions, and suggest the method is suitable for further use in clinical practice. Hepatic iron quantification by AAS is validated by the experiments performed in the present study.