Development of an ultra-high performance liquid chromatography analytical methodology for the profiling of olive (Olea europaea L.) pulp proteins

Ultra-high performance liquid chromatography (UHPLC) constitutes an interesting proposal to speed protein separations but it is almost not explored. In this work UHPLC is proposed, for the first time, to separate olive pulp proteins. An important difficulty in the analysis of proteins is related to their extraction. The difficulty in the extraction of proteins from the olive pulp is derived from its high content in lipids and phenolic compounds. Eight different methods for the extraction of pulp proteins were designed and evaluated. The optimized extraction procedure consisted of a cleaning step to remove interfering compounds, followed by the extraction of proteins with a Tris–HCl buffer containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT), precipitation of proteins with acetone, and solubilization in the Tris–HCl buffer. This methodology yielded the most successful isolation of pulp proteins and enabled the optimization of a UHPLC methodology for their separation. The method was applied to the profiling of olive pulp proteins from different olive cultivars observing in all cases a protein that had never been described before.     

Quantitative analysis and evaluation of the solubility of hydrophobic proteins recovered from brain, heart and urine using UV-visible spectrophotometry

There is a need for a simple method that can directly quantify hydrophobic proteins. UV-visible spectrophotometry was applied in the present study for this purpose. Absorbance at λ=280 nm (A ₂₈₀) was detected for both Escherichia coli membrane proteins and bovine serum albumin, whereas absorbance at λ=620 nm (A ₆₂₀) was only detected for E. coli membrane proteins. The A ₆₂₀ values of the brain samples were greater than those of heart samples when equal concentrations were used, regardless of the type of solubilizing agent employed. Because hydrophobic proteins tend to form colloidal microparticles in solution, we also applied UV-visible spectrophotometry to evaluate the efficacies of different extraction protocols for solubilizing hydrophobic proteins. For brain protein extraction, the highest A ₆₂₀ was observed in samples recovered using Tris, whereas the lowest was from samples recovered using SDS. Solubilizing brain tissue with 0.25% SDS (above the CMC) gave a lower A ₆₂₀ than extraction with 0.025% SDS (below the CMC). Addition of 0.25% SDS to samples recovered with Triton caused A ₆₂₀ to drop. A ₆₂₀ could also be used to distinguish between the hydrophobic fractions (pellets) of brain and urine proteins and their hydrophilic fractions (supernatants) prefractionated using high-speed centrifugation. Additionally, an A ₆₂₀/A ₂₈₀ ratio exceeding 0.12 appears to denote highly hydrophobic samples. Our data suggest that direct UV-visible spectrophotometry can be used as a simple method to quantify and evaluate the solubilities of hydrophobic proteins.     

Solubilization and Hydrolysis of Ovotransferrin Solubilization of α-chymotrypsin. Enthalpy changes for three processes

At 298.15 K, the solubilization of hen ovotransferrin at buffered pH 7.8 (0.08 M Tris⋅HCl buffer, containing 0.1 M CaCl₂) and the solubilization of α-chymotrypsin (from bovine pancreas) at non-buffered pH 3.0 (0.001 M HCl) both resulted in large exothermic reactions, being the apparent ΔHs –2485 in the first case and –780.1 kJ mol^–1 in the second case, respectively. By contrast, the complete hydrolysis of ovotransferrin (pH 7.8) achieved by using a-chymotrypsin (pH 3.0) gave an endothermic reaction with ΔH=+31.84 kJ mol^–1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protein-like copolymers: Computer simulation

The model ofAB copolymers with a “protein-like” primary sequence was developed. This type of copolymers was obtained in a computer experiment. First, the conformation of a collapsed dense homopolymer globule was generated and then, based on this conformation, the primaryAB sequence was determied by denoting the monomeric units located near the surface of the globule as unitsA and those constituting the core of the globule as unitsB. After that, the primary structure of the chain was fixed, and different interaction potentials for theA andB units were introduced. Drawing an analogy of this model to aqueous solutions of globular proteins,A units were interpreted as hydrophilic, andB units were regarded as hydrophobic. By means of Monte Carlo simulation using the bond fluctuation model, the coli—globule transition in “protein-like”AB copolymer, induced by an increase in the attraction between the hydrophobicB units, was studied. The coil—globule transition in a copolymer with the “protein-like” primary sequence occurs at a higher temperature and has higher rate and is sharper than that in a random copolymer with the sameA/B composition and in a random block copolymer with the sameA/B composition and the same “degree of blockiness”. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 884–889, May, 1998.     

On-line coupling of physiologically relevant bioaccessibility testing to inductively coupled plasma spectrometry: Proof of concept for fast assessment of gastrointestinal bioaccessibility of micronutrients from soybeans

In-vitro physiologically relevant gastrointestinal extraction based on the validated Unified BARGE Method (UBM) is in this work hyphenated to inductively coupled plasma optical emission spectrometry in a batch-flow configuration for real-time monitoring of oral bioaccessibility assays with high temporal resolution. A fully automated flow analyzer is designed to foster in-line filtration of gastrointestinal extracts at predefined times (≤15 min) followed by on-line multi-elemental analysis of bioaccessible micro-nutrients, viz., Cu, Fe and Mn, in well-defined volumes of extracts (300 μL) of transgenic and non-transgenic soybean seeds taken as model samples.     

Electrochemical behavior of nicotinamide using carbon paste electrode modified with macrocyclic compounds

The electrochemical behavior of nicotinamide was studied at a carbon paste electrode and the electrodes modified with macrocyclic compounds using voltammetric and impedance measurements. The electrodes so formed were able to bind nicotinamide ions chemically and gave better voltammetric responses than the unmodified ones. The macrocycles used as modifiers for the electrode preparation were 18-crown-6, dicyclohexano-18-crown-6, dibenzo-18-crown-6, 7,16-dibenzyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane, 1,4,7,10,13,16-hexathiacyclooctadecane (Hexathia), 1,4,7,10-tetratosyl-1,4,7,10-tetraazacyclododecane, 1,4,8,11-tetraazacyclooctadecane, c-Methylcalix[4]resorcenarene and calix[8]arene. Among these macrocyclic modified electrodes, hexathia showed more affinity towards nicotinamide and a 2.3-fold increase in voltammetric signal was obtained. Impedance measurement was used to confirm this enhancement observed on modified electrode. This increase in anodic peak current was then used for finding linear working range, which was 0.1–500 μg mL⁻¹ with a detection limit of 0.03 μg mL⁻¹ by DPV. Interference from other vitamins like thiamine HCl (Vit. B₁), riboflavin (Vit. B₂), pyridoxine HCl (Vit. B₆) cynocobamine (Vit. B₁₂), para-aminobenzoic acid (PABA) and ascorbic acid (Vit. C) was also studied. The modified electrode could be used for the simultaneous determination of riboflavin, nicotinamide and pyridoxine HCl. It has also been utilized for the analysis of nicotinamide in pharmaceutical preparations.     

Cesium exchange reaction on natural and modified clinoptilolite zeolites

Cesium cation exchange reaction with K, Na, Ca and Mg ions on natural and modified clinoptilolite has been studied. Batch cation-exchange experiments were performed by placing 0.5 g of clinoptilolite into 10 ml or 20 ml of 1·10⁻³M CsCl solution for differing times. Two type deposits of clinoptilolite zeolites from, Nižny Hrabovec (NH), Slovakia and Metaxades (MX), Greece were used for ion-exchange study. The distribution coefficient (K _d ) and sorption capacity (Γ) were evaluated. For the determination of K, Na, Ca and Mg isotachophoresis method, the most common cations in exchange reaction was used. Cesium sorption was studied using ¹³⁷Cs tracer and measured by γ-spectrometry.     

Conductance of tris(hydroxymethyl)-aminomethane hydrochloride (Tris·HCl) in water at 25 and 37°C

The conductances of aqueous solutions of tris(hydroxymethyl)aminomethane hydrochloride (Tris·HCl) at 25°C and 37°C have been measured. The concentration of salt varied from 4×10^–4 to 1.6×10^–2 mole-dm^–3. The data were analyzed by the full Pitts equation which yielded the following parameters: at 25°C, ° = 106.07 S-cm²-mole^–1, K_A=1.01; at 37°C, ° = 106.07 S-cm²-mole^–1, K_A=0.99. These values suggest that Tris·HCl is essentially completely dissociated in water. The mobility of the Tris·H⁺ ion was found to be considerably smaller than that of the alkali metal cations. This result is consistent with abnormal liquid-junction potentials for Tris buffer solutions at 25 and 37°C.     

Sample preparation by in-gel digestion for mass spectrometry-based proteomics

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “in-gel digestion” has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches. Figure Classical in-gel digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel (1). CBB molecules (blue circles) bound to the protein are released by iterative incubation in a buffered organic solvent system (2). To increase digestion efficiency and sequence coverage proteins are reduced (3) and alkylated (4). Proteins are subsequently digested with proteolytic enzymes (scissors symbols), typically trypsin (5). Trypsin cleaves at the amino acid residues arginine (R) and lysine (K). The resulting peptides (A, B, and C) are extracted from the polyacrylamide matrix (6). The peptide solution can be further purified for analysis by mass spectrometry (Section “Concentration and desalting of peptides”)     

Evaluation of gel electrophoresis techniques and laser ablation–inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton

The determination of metal-binding proteins in plankton is important because of their involvement in photosynthesis, which is fundamental to the biogeochemical cycle of the oceans and other ecosystems. We have elaborated a new strategy for screening of Cu and Zn-containing proteins in plankton on the basis of separation of proteins by use of Blue-Native PAGE (BN-PAGE), which entails use of a non-denaturing Tris–tricine system and detection of metals in the proteins by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS). For comparison, denaturing PAGE based on Tris–glycine and Tris–tricine systems and Anodic-Native PAGE have also been investigated. A large number of protein bands with MW between 20 and 75 kDa were obtained by use of Tris–glycine PAGE but detection of metals by LA–ICP–MS was unsuccessful because of loss of metals from the proteins during the separation process. Different protein extraction, purification, and preconcentration methods were evaluated, focussing on both issues—achieving the best extraction and characterization of the proteins while maintaining the integrity of metal–protein binding in the plankton sample. Use of 25 mmol?L^?1 Tris–HCl and a protease inhibitor as extraction buffer with subsequent ultrafiltration and acetone precipitation was the most efficient means of sample preparation. Two Cu and Zn proteins were detected, a protein band corresponding to a MW of 60 kDa and another poorly resolved band with a MW between 15 and 35 kDa.     

Reciprocity relation in scattering by media subjected to an external magnetic field

Summary Krishnan's reciprocity theorem in colloid optics,ϱ _u=1+l/ϱ_h/1+1/ϱ _v is generalised for the case when the scattering medium is subjected to an external orienting field. It is shown theoretically that a general relation of the typeI _B ^A =I′ _A ^B results in this case, whereI _B ^A is the intensity of the component of the scattered light having its electric vector inclined at an angleB to the vertical with the incident light polarised at an angleA to the vertical, the external field direction being parallel to the incident beam.I′ _A ^B is the corresponding intensity with the magnetic field parallel of the scattered ray. Experimental verification of the above generalisation is also given.

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Zusammenfassung Krishnans Reziprozit?tstheoremϱ _u=1+1/ϱ _h/1+1/ϱ _v wird für den Fall verallgemeinert, da\ das streuende Medium einem ?u\eren orientierenden Feld unterliegt. Es l?\t sich theoretisch zeigen, da\ in diesem Fall eine allgemeine Beziehung des TypsI _B ^A =I′ _A ^B resultiert, in derI _B ^A die Intensit?t der Komponente des Streulichts mit dem elektrischen Vektor um den WinkelB zur Vertikalen geneigt bei einfallendem Licht um einen WinkelA zur Vertikalen polarisiert angibt, und das ?u\ere Feld parallel zum einfallenden Strahl liegt.I′ _A ^B ist die entsprechende Intensit?t mit dem Magnetfeld parallel zum gestreuten Strahl. Eine experimentelle Best?tigung der genannten Verallgemeinerung wird gegeben.

     

Chemical analysis by inductively coupled plasma atomic emission spectrometry of semiconducting ceramics of barium titanate doped with various metal oxides

The Ba and Ti macroconstituents as well as the impurities and dopants content (Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Nb, Ni, P, Pb, Si, Sr, W, Zn and Zr) in a dense (> 98% theoretical) barium titanate sample have been determined by inductively coupled plasma-atomic emission spectrometry after one of these decomposition routes: (a) decomposition with HCl in a PTFE-lined pressure vessel, (b) fusion with Na₂CO₃ in a platinum crucible, and (c) fusion with Li₂B₄O₇ in a graphite crucible. Matrix effects were taken into account. Detection limits for minors and trace elements were determined. High sensitivity and good precision were attained.Presented in part at the 1989 European Winter Conference on Plasma Spectrochemistry, Reutte, Austria     

Interaction of <Emphasis Type="Italic">Moringa oleifera</Emphasis> seed lectin with humic acid

The aim of this work was to characterise the affinity of protein preparations from Moringa oleifera seeds, specifically extract (seeds homogenised with 0.15 M NaCl), fraction (extract precipitated with 390 mg mL⁻¹ of ammonium sulphate) and cMoL (coagulant M. oleifera lectin) to bind humic acids using a haemagglutinating activity assay with rabbit erythrocytes and a radial diffusion assay in agarose gel. Specific haemagglutinating activity (SHA) decreased by 94 % for the extract and cMoL and by 50 % for the fraction in the presence of humic acid. Precipitation bands were observed in the diffusion gel. Both results suggested humic acid-cMoL binding. Carbohydrates, potassium, and calcium ions and pH affected the SHA of cMoL. As an example of application, cMoL was immobilised on a column packed with sepharose receiving 20 mg mL⁻¹ of carbon humic acid solution, 30 mg of humic acid per gram of support was removed. This result suggested that protein preparations might be used in water treatment to remove humic acids.     

ESR characterization and kinetics of the enzymatically obtained M(II)-dobutamine-o-semiquinone system

Dobutamine is a synthetic catecholamine used in the treatment of different cardiac pathologies associated with organic heart diseases, myocardial infarction and cardiac surgery. The ESR spectra of M²⁺-o-semiquinone radicals, obtained by the oxidation of dobutamine HCl with horseradish peroxidase and hydrogea peroxide in the presence of several bivalent metal ions of IIA (Mg, Ca, Sr, Ba) and IIB (Zn, Cd) groups, have been reported.     

Ubiquinol formation in isolated photosynthetic reaction centres monitored by time-resolved differential FTIR in combination with 2D correlation spectroscopy and multivariate curve resolution

Two-dimensional correlation analysis was carried out in combination with multivariate curve resolution–alternating least squares (MCR-ALS) to analyse time-resolved infrared (IR) difference spectra probing photo-induced ubiquinol formation in detergent-isolated reaction centres from Rhodobacter sphaeroides. The dynamic 2D IR correlation spectra have not only allowed the determination of the concomitance or non-concomitance of different chemical events through known marker bands but also have helped identify new vibrational bands related to the complex series of photochemical and redox reactions. In particular, a strong positive band located at 1565 cm⁻¹ was found to be synchronous with the process of ubiquinol formation. In addition, a tailored MCR-ALS analysis was performed using a priori chemical knowledge of the system, in particular including the pure spectrum of one species obtained from an external measurement. Enhancing the MCR-ALS performance in this way in time-dependent processes is relevant, especially when other essential pieces of information, such as kinetic models, are unavailable. The results give evidence of four independent spectral contributions. Three of them show marker bands for a monoelectronic reduction of the primary quinone Q_A (Q_A⁻/Q_A transition, first contribution), for a monoelectronic reduction of a secondary quinone Q_B (Q_B⁻/Q_B transition, second contribution) and for ubiquinol formation (third contribution). The results obtained also confirm that a key rate-limiting factor is the slow ubiquinone and ubiquinol exchange among micelles, which strongly influences the kinetic profiles of the involved species.     

Acid leaching purification and neutron activation analysis of high purity silicas

The effects of acids on the removal of impurity from 2N grade silica have been studied using five leaching acids: 0.2 M-oxalic acid (pH 1.5 and 2.5), c-aqua regia, 2.5%—HCl/HF, and 1%—HNO₃/HF. The presence of 39 impurities in the 2N grade silica and the reference material (RM, 5N grade silica) were investigated by neutron activation analysis (NAA), X-ray fluorescence (XRF), and inductively coupled plasma mass spectrometry (ICP-MS) methods. Major impurities of the 2N silica were Al, K, Fe, Na, Ti, Ca, Mg and P. The fractions of the eight major impurities were 99.2% and 90.9% of total impurity in the 2N and RM silica, respectively. Among the leaching acids, almost all of the major impurities were removed effectively by the 2.5% HCl/HF leaching acid. All the major impurities, except for phosphorous, as well as 21 minor and trace impurities could be determined by the NAA.     

Study of the protein-bound fraction of calcium,iron, magnesium and zinc in bovine milk

Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry (ICPOES). Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g l⁻¹ trichloroacetic acid (TCA), and an enzymatic protein hydrolysis using 50 g l⁻¹ pepsin (PEP) solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions.     

Application of elemental bioimaging using laser ablation ICP-MS in forest pathology: distribution of elements in the bark of Picea sitchensis following wounding

Element distribution in the bark of two 20-year-old clones of Picea sitchensis following wounding was studied using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Bark was sampled at 0, 3, and 43 days after wounding and analysed using a focused Nd:YAG laser (266 nm). Intensities of ¹³ C, ²⁵Mg, ²⁷Al, ³¹P, ³²S, ³⁹K, ⁴⁸Ca, ⁵⁵Mn, ⁵⁷Fe, ⁶³Cu and ⁶⁴Zn were measured by ICP-MS to study elemental distribution across the bark samples during the wound repair process. A clear accumulation of Mg, P and K at the boundary zone between the lesion and healthy tissue was detected in the wounded samples and was more distinctive at 43 than at 3 days after treatment. This zone of accumulation mapped onto the position of formation of the ligno-suberised boundary zone and differentiation of the wound periderm. These accumulations suggest major roles for Mg, P and K in the non-specific response of Sitka spruce both to wounding, possibly as co-factors to enzymes and energy utilisation. The LA-ICP-MS method developed in this work proved useful to study spatial element distribution across bark samples and has great potential for applications in other areas of plant pathology research.     

Thermal studies on protein isolates of white lupin seeds (<Emphasis Type="Italic">Lupinus albus</Emphasis>)

This study used TG, DSC, and SDS-PAGE techniques to study protein isolates (PIs) in the powder form obtained from lupin seeds flour Lupinus albus. Different methods of preparing PIs were tested, resulting in final products that were different only in relation to the yield and protein content. The results of the protein analysis by SDS-PAGE showed that the same protein fractions were present in the lupin seeds and in the obtained PIs. This result shows that the process of extraction was not damaging to the composition of the original protein. On the other hand, the results of the thermal analysis (DSC and TG–DTG curves) obtained for the different PIs, led to the detection of changes in the protein conformation through the ΔH values, which in general decreased with increasing values of pH and ionic strength in the experimental conditions of extraction.     

Viscosity of Aqueous Solutions of Lithium,Sodium, Potassium,Rubidium and Caesium Cyclohexylsulfamates from 293.15 to 323.15 K

The viscosities of aqueous solutions of lithium, sodium, potassium, rubidium and caesium cyclohexylsulfamates were measured at 293.15, 298.15, 303.15, 313.15 and 323.15 K. The relative viscosity data were analyzed and interpreted in terms of the Kaminsky equation, η _r=1+Ac ^1/2+Bc+Dc ². The viscosity A-coefficient was calculated from the Falkenhagen-Dole theory. The viscosity B-coefficients are positive and relatively large. Their temperature coefficient ∂ B/∂ T is negative or near zero for lithium and sodium salts whereas for potassium, rubidium and caesium salts it is positive. The viscosity D-coefficient is positive. This was explained by the size of the ions, structural solute–solute interactions, hydrodynamic effect, and by higher terms of the long-range Debye-Hückel type of forces. From the viscosity B-coefficients the thermodynamic functions of activation of viscous flow were calculated. The limiting partial molar Gibbs energy of activation of viscous flow of the solute was divided into contributions due to solvent molecules and the solute in the transition state. The activation energy of the solvent molecules was calculated using the limiting Gibbs energy of activation for the conductance of the solute ions. The activation energy of the solvent molecules was then discussed in terms of the nature of the alkali-metal ions and their influence on the structure of water. The limiting activation entropy and enthalpy of the solute for activation of viscous flow were interpreted by ion-solvent bond formation or breaking in the transition state of the solvent. The hydration numbers of the investigated electrolytes were calculated from the specific viscosity of the solutions.