Surface Activity Of Proteins Research Articles

Characterization of inflammation in a rat model of acute lung injury after repeated pulmonary lavage

ABSTRACTAim of the study: Repeated pulmonary lavage allows to reliably reproduce failure of gas exchange and major histological findings of acute lung injury (ALI). However, because the capacity of pulmonary lavage to induce pulmonary inflammation is not well established in rodents, this study aims to characterize the induction of pulmonary inflammation in a rat model of ALI.Materials and Methods: Male adult rats were divided into a treatment group (n = 9) that received pulmonary lavage with consecutive mechanical ventilation, and a control group that received mechanical ventilation only (n = 9). Arterial blood gas analyses were performed every 30 min throughout the study. Pressure-volume curves, and lung tissue and plasma samples, were obtained at 240 min after the start of mechanical ventilation. Protein content and surface activity of bronchoalveolar lavage fluid was assessed. Transcriptional and translational regulation of pro- and anti-inflammatory cytokines IL-1β, TNF-α, IL-6, and IL-10 was determined in lungs and plasma. Markers of cellular stress were measured in lung tissue.Results: Pulmonary lavage significantly decreased lung compliance, induced hypoxia and hypercapnia, and mediated respiratory acidosis. Protein content of lavage fluid was significantly increased and contained washed out surfactant. Expression of IL-1β, TNF-α, and IL-6 mRNA and protein expression of IL-1β and TNF-α was significantly induced in lavaged lungs, without spillover into the systemic circulation. Markers of cellular stress were significantly upregulated in lavaged lungs.Conclusions: This model of ALI applied in rats can induce pulmonary inflammation. The model might be used to develop therapeutic strategies that target pulmonary inflammation in ALI.

Competitive Adsorption of Lecithin and Saliva at the O/W Interface in Relation to the Oral Processing of Lipid Continuous Foods

This research is relevant to oral processing of lipid continuous foods. During this first step of food digestion, lipid continuous foods such as chocolate or margarine phase invert into oil-in-water emulsions stimulated through the mechanical action of tongue and teeth in combination with the change in temperature and the high surface activity of salivary proteins. These are hypothesised to stabilise the newly formed interface in competition with surfactants or surface active molecules released from the food if present. Here competitive adsorption between mechanically stimulated human whole saliva (HWS) and lecithin dissolved in sunflower oil freed of interfacially active contaminants was investigated in-vitro using a pendant drop tensiometer for dynamic interfacial tension and interfacial rheological measurements. Initially, it was validated that the interfacial properties of HWS samples remained unaffected by frozen storage at −80 °C during 6 weeks. Protein concentration affected the absolute values of interfacial tension and in particular the dilatational elastic modulus. Competitive adsorption studies revealed a mixed interface and it follows that emulsion stabilisation during oral processing involves both salivary proteins and lecithin present in the oil phase.

Anticancer Activity from Gold-alpha-Lactalbumin Nanoconstructs?

Human α-Lactalbumin made lethal to tumor cells (HAMLET), and its bovine analogue BAMLET, have in the recent years shown promising results in cancer therapy. HAMLET contains several oleic acids which in turn stabilize a partially unfolded conformational state of the protein, inducing a higher surface activity compared to the native protein, and likely play a role in its cancer-detrimental function as well. Herein, we report the formation of gold-bovine α-lactalbumin nanoconstructs (Au–LA NCs) displaying conformational changes in the native protein and its concomitant ability to reduce HeLa cell viability approaching that of BAMLET. Modification of LA with gold was achieved via two synthesis protocols; (i) utilizing the intrinsic reduction potential of LA (Au–LAint) or (ii) the addition of an extrinsic reducing agent (Au–LAext). The gold–protein nanostructures formed were investigated using a palette of analytical probes including AFM, TEM, ζ-potential, UV–vis, circular dichroism, and fluorescence (steady-state and time-resolved). Toxicity toward HeLa cells was studied using a trypan blue assay and benchmarked against BAMLET. Whereas constructs from both synthetic protocols employed result in conformational changes of the protein and altered surface activity compared to the native protein, Au–LAint was found to display lipid-specific interaction and severe disruption of lipid monolayers. Au–LAint also revealed toxicity toward HeLa-cells comparable to that of BAMLET. The results imply that formation of Au–LA nanoconstructs could be a new route to making HAMLET-like materials, while also imparting a built-in optical probe from fluorescent or plasmonic gold species.

Inhibition of Glycogen Synthase Kinase-3 increases NKG2D Ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in Multiple Myeloma cells: role of STAT3.

Event Abstract Back to Event Inhibition of Glycogen Synthase Kinase-3 increases NKG2D Ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in Multiple Myeloma cells: role of STAT3. Cinzia Fionda1*, Giulia Malgarini1, Alessandra Soriani1, Alessandra Zingoni1, Francesca Cecere1, Marialuisa Iannitto1, Maria Rosaria Ricciardi2, Maria Teresa Petrucci2, Angela Santoni1 and Marco Cippitelli1 1 University of Rome "Sapienza", Department of Molecular Medicine, Italy 2 Sapienza, Department of Ematology, Italy Engagement of NKG2D and DNAM-1 receptors with their ligands on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the Glycogen Synthase Kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in human MM (Multiple Myeloma) cells. GSK3 is a serine-threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 is able to up-regulate MICA protein surface, mRNA expression and promoter activity in MM cells without significant effects on the expression of MICB and DNAM-1 ligand PVR/CD155. Moreover, exposure to GSK-3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and trigger NK cell-mediated cytotoxicity. We could exclude that two transcription factors specifically inhibited by active GSK3, β-catenin or Heat Shock Factor-1 (HSF-1), are involved in the up-regulation of MICA expression by using RNA-interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a significant downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr705 phosphorylation and in vivo binding of STAT3 on MICA promoter are reduced by drugs targeting GSK3; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional mechanism supporting the anti-myeloma activity of GSK3 inhibitors, and suggest their possible immunotherapeutic value. Keywords: NKG2D ligands, ; GSK3, Multiple Myeloma, NK, STAT3 Transcription Factor Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Fionda C, Malgarini G, Soriani A, Zingoni A, Cecere F, Iannitto M, Ricciardi M, Petrucci M, Santoni A and Cippitelli M (2013). Inhibition of Glycogen Synthase Kinase-3 increases NKG2D Ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in Multiple Myeloma cells: role of STAT3.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00214 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Cinzia Fionda, University of Rome "Sapienza", Department of Molecular Medicine, Rome, 00161, Italy, cinzia.fionda@uniroma1.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Cinzia Fionda Giulia Malgarini Alessandra Soriani Alessandra Zingoni Francesca Cecere Marialuisa Iannitto Maria Rosaria Ricciardi Maria Teresa Petrucci Angela Santoni Marco Cippitelli Google Cinzia Fionda Giulia Malgarini Alessandra Soriani Alessandra Zingoni Francesca Cecere Marialuisa Iannitto Maria Rosaria Ricciardi Maria Teresa Petrucci Angela Santoni Marco Cippitelli Google Scholar Cinzia Fionda Giulia Malgarini Alessandra Soriani Alessandra Zingoni Francesca Cecere Marialuisa Iannitto Maria Rosaria Ricciardi Maria Teresa Petrucci Angela Santoni Marco Cippitelli PubMed Cinzia Fionda Giulia Malgarini Alessandra Soriani Alessandra Zingoni Francesca Cecere Marialuisa Iannitto Maria Rosaria Ricciardi Maria Teresa Petrucci Angela Santoni Marco Cippitelli Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces

One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested (“mAb-1”) showed highest surface loading to silica at pH 7.4 (~12 mg/m2), with lower surface loading at pH 5.5 (~5.5 mg/m2, further from its pI of 8.99) and to hydrophobized silica (~2 mg/m2). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50–5000 mg/L, pH 5.5, and 50–2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO2 surface at near saturation with an end-on” orientation, while the outer mAb-1 layer was sparse and molecules had a “side-on” orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO2 surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding.

Investigation of Relationship between Surface Tension of Feed Solution Containing Various Proteins and Surface Composition and Morphology of Powder Particles

The surface tension of freshly created food protein powder isolates was measured in aqueous solutions as a function of concentration, hydrolysis, and temperature. The surface tension of the solutions was measured immediately to best predict their surface-active behavior in a spray-drying scenario, where instantaneous values are more relevant than equilibrium surface tension measurements. Whole whey protein, hydrolyzed whey proteins (degrees of hydrolysis of 4, 9.5, 12, 17, and 20.2%), soy protein, pea protein isolates, and gelatin powders were diluted in a range of concentrations (0.04–2 g/L) and their surface tension values were reported at 23 ± 1°C. It was found that at higher concentrations hydrolyzed whey proteins at degrees of hydrolysis of 9.5 and 12%, and soy protein isolates in particular, showed excellent surface activity (shown through a decrease in surface tension) compared to nonhydrolyzed whey protein and gelatin. When comparing the influence of the degree of hydrolysis of whey proteins, the reverse was observed at lower concentrations (0.04–0.1 g/L), with the nonhydrolyzed whey protein reducing surface tension values more effectively than their hydrolyzed counterparts. Additionally, the protein solutions (2 g/L) were maintained at higher temperatures of 40, 50, and 60°C and the surface tension values were measured. There was a general improvement of surface activity of proteins indicated by the reduced surface tension of solutions at these temperatures compared with the pure water values. The protein solutions were also spray dried with maltodextrin (MD30) and the powder particle surface composition and structures were analyzed via X-ray photoelectron spectroscopy and scanning electron micrography. There was a trend of correlation between the surface activities of protein in solution with that of the surface composition of protein found on the powder particles. However, there were morphological indicators that corresponded well to the amount of protein present on the surface.

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.

The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Coronary Artery Endothelial Cells

The discovery of the role of P2Y(12) receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y(2) and P2Y(11) receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y(2) receptor, but not by NF-157, a P2Y(11)-selective antagonist, suggesting a role for the P2Y(2) receptor. In addition, P2Y(2) receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca(2+) pathway did not affect P2Y(2) receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y(2) receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.

Preparation of Water-Soluble Soy Protein-Based Surfactants by Oxidizing Disulfide Bonds

In this paper, the correlations between the cleavage degree of disulfide bonds and soy protein surface activity had been studied in order to show the surface activity of soy protein. The disulfide bonds of soy protein were oxidized to sulfonic groups by performic acid. The distribution of polar and nonpolar groups, and the molecular structure of soy protein were changed because of the oxidation damage of disulfide bonds, and these changes led to changes in surface activity of soy protein. The results showed that the emulsifying property of soy protein was improved by oxidizing the disulfide bonds of protein compared with that of natural soy protein. The change of soy protein emulsifying property is closely related with the degree of the disulfide bond oxidation damage,and that was also an effective way to prepare the protein-based surfactant.

Surface accumulation of milk proteins and milk protein hydrolysates at the air–water interface on a time-scale relevant for spray-drying

Accumulation of surface-active compounds may significantly alter the surface composition of food powders compared to the bulk composition of the product and therefore affects the functional properties. Through modification of a commercial contact angle meter surface accumulation of milk proteins and protein hydrolysates could be analysed via surface tension measurements in a time interval relevant for atomisation of emulsions during microencapsulation. The different caseins showed a slower surface occupation compared to industrially processed sodium caseinate and casein hydrolysate with β-casein being more surface-active than α- and κ-casein. In a similar manner, β-lactoglobulin showed a lower surface activity than whey protein isolate and whey protein hydrolysate. It is concluded that molecular weight profile plays an important role for the surface activity of milk proteins, but other factors like surface hydrophobicity, number of ionisable groups and state of aggregation also need to be considered.

Mass Spectrometry of Laser-Initiated Carbene Reactions for Protein Topographic Analysis

We report a protein labeling method using nonselective carbene reactions of sufficiently high efficiency to permit detection by mass spectrometric methods. The approach uses a diazirine-modified amino acid (l-2-amino-4,4'-azipentanoic acid, "photoleucine") as a label source, which is converted to a highly reactive carbene by pulsed laser photolysis at 355 nm. Labeling of standard proteins and peptides (CaM, Mb, M13) was achieved with yields up to 390-fold higher than previous studies using methylene. Carbene labeling is sensitive to changes in protein topography brought about by conformational change and ligand binding. The modification of apo-CaM was 45 ± 7% higher than that of holo-CaM. Modification of the CaM-M13 complex reflected a 39 ± 1% reduction in labeling for bound holo-CaM relative to free holo-CaM. Labeling yield is independent of protein concentration over approximately 2 orders of magnitude but is weakly dependent on the presence of other chromophores in a photon-limited apparatus. The current configuration required 2 min of irradiation for full reagent conversion; however, it is shown that comparable yields can be achieved with a single high-energy laser pulse (>100 mJ/pulse, <10 ns), offering a labeling method with high temporal resolution. We suggest a mechanism of labeling governed by limited carbene diffusion and the protein surface activity of the diazirine precursor. This surface activity is speculated to return a measure of selectivity relative to methylene labeling, which ultimately may be tunable.

Enhancement of emulsifying properties of whey proteins by controlling spray-drying parameters

Whey protein concentrate is the main source of globular proteins in food products which are principally used as emulsifying, foaming and gelling ingredients. These whey proteins are commonly used in powder form obtained by a spray-drying process. It is well known that β-lactoglobulin, the major protein component in whey, is greatly affected by heat treatments, with consequences on its adsorption properties at fluid–fluid interfaces. This study concerned four whey protein powders obtained using spray-drying at four different air inlet temperatures (from 170 to 260 °C), leading to different levels of protein solubility, denaturation and end-use properties. After evaluation of the protein denaturation by HPLC, the emulsifying properties were studied through particle size parameters and rheological properties in relation with spray-drying parameters. Our results indicated that oil-in-water emulsions, stabilized by 5% (w/w) protein samples, exhibited a shear-thinning flow behaviour, and the harsher the spray-drying conditions (the higher the protein denaturation), the less viscous were the emulsions. The apparent viscosity of emulsions measured at 20 °C and 50 s −1 shear rate was around 0.08 Pa s when containing whey proteins before drying, and around 0.05–0.018 Pa s after drying at air inlet temperatures from 170 to 260 °C. These differences in emulsion rheological properties were related to particle size effects, in regards to analysis of particle size distributions which showed a finer emulsion according to spray-drying intensity. Our results will be presented and discussed in terms of optimization of spray-drying process relative to globular protein surface activity.

Equilibrium of Adsorption of Mixed Milk Protein/Surfactant Solutions at the Water/Air Interface

Ellipsometry and surface profile analysis tensiometry were used to study and compare the adsorption behavior of beta-lactoglobulin (BLG)/C10DMPO, beta-casein (BCS)/C10DMPO and BCS/C12DMPO mixtures at the air/solution interface. The adsorption from protein/surfactant mixed solutions is of competitive nature. The obtained adsorption isotherms suggest a gradual replacement of the protein molecules at the interface with increasing surfactant concentration for all studied mixed systems. The thickness, refractive index, and the adsorbed amount of the respective adsorption layers, determined by ellipsometry, decrease monotonically and reach values close to those for a surface covered only by surfactant molecules, indicating the absence of proteins from a certain surfactant concentration on. These results correlate with the surface tension data. A continuous increase of adsorption layer thickness was observed up to this concentration, caused by the desorption of segments of the protein and transforming the thin surface layer into a rather diffuse and thick one. Replacement and structural changes of the protein molecules are discussed in terms of protein structure and surface activity of surfactant molecules. Theoretical models derived recently were used for the quantitative description of the equilibrium state of the mixed surface layers.

Amiodarone Inhibits Arterial Thrombus Formation and Tissue Factor Translation

In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation. Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation. Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. Guggulsterone inhibited TNF-alpha-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-alpha-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-alpha-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

Surface and interfacial properties of water-soluble wheat proteins

Surface and interfacial properties of water-soluble wheat proteins were investigated and compared with six reference proteins (bovine serum albumin, ovalbumin, β-lactoglobulin, trypsin, cytochrom C and β-amylase). Albumins extracted from wheat flour were separated by the free solution isoelectric focusing. The surface activity at the air/water, dodecane/water interfaces and dilatational rheological behaviour of the adsorbed layers was determined by pendant drop technique. Considerably high surface activity of wheat proteins was found at both interfaces exceeding the corresponding values of most of the reference proteins. Exceptionally, low dilatational moduli (typically ɛ < 10 mN/m) were obtained for wheat fractions in the continuous and the stepwise compression experiments with no age effect (1–20 min) and almost no relaxation. Surface/interfacial activity and rheological properties observed imply that water-soluble wheat proteins are generally characterized by strong hydrophobicity and more flexible molecular structure than the reference proteins.

Molecular Details of Ovalbumin−Pectin Complexes at the Air/Water Interface: A Spectroscopic Study

To stabilize air-water interfaces, as in foams, the adsorption of surface-active components is a prerequisite. An approach to controlling the surface activity of proteins is noncovalent complex formation with a polyelectrolyte in the bulk phase. The molecular properties of egg white ovalbumin in a complex with pectin in the bulk solution and at air/water interfaces were studied using drop tensiometry (ADT) and time-resolved fluorescence anisotropy techniques. The complex formation of ovalbumin with pectin in the bulk resulted in the formation of a compact structure with a different spatial arrangement depending on the protein/pectin ratio. Complex formation did not provide an altered protein structure, whereas the conformational stability was slightly increased in the complex. In excess pectin, an overall condensed complex structure is formed, whereas at limited pectin concentrations the structure of the complex is more "segmental". The characteristics of these structures did not depend on pH in the 7.0 to 4.5 regime. Interaction with pectin in the bulk solution resulted in a significantly slower adsorption of the protein to the air/water interface. The limited mobility of the protein at the interface was found for both ovalbumin and ovalbumin-pectin complexes. From both the rotational dynamics and total fluorescence properties of the protein in the absence and presence of pectin, it was suggested that the complex does not dissociate at the interface. Ovalbumin in the complex retains its initial "aqueous" microenvironment at the interface, whereas in the absence of pectin the microenvironment of the protein changed to a more nonpolar one. This work illustrates a more general property of polyelectrolytes, namely, the ability to retain a protein in its microenvironment. Insight into this property provides a new tool for better control of the surface activity of complex biopolymer systems.

Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV–visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid–air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid–air interface of 4-μl drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid–air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid–air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins—chicken egg albumin, bovine serum albumin, human serum albumin, α-lactalbumin (α-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, α-chymotrypsinogen A, β-lactoglobulin (β-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase—is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0–8, 0–6, and 0–5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air–liquid interface of two proteins, α-Lac and β-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.

High-Throughput Screening of Protein Surface Activity via Flow Injection Analysis-pH Gradient-Dynamic Surface Tension Detection

Using flow injection analysis (FIA), a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). This instrumental system was developed as a high-throughput method for the screening of protein surface activity at the air/liquid interface as a function of pH. This method utilizes the continuous flow, drop-based dynamic surface tension detector in combination with flow injection sample introduction and blending of a steady-state concentration of protein sample with a pH gradient ranging from pH 2.0 to pH 11.5. Dynamic surface tension is measured through the differential pressure across the air/liquid interface of repeatedly growing and detaching drops. Continuous surface tension measurement is achieved for each eluting drop of 2-s length (2 muL), providing insight into both the kinetic and thermodynamic behaviors of molecular orientation processes at the liquid/air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is collected as a function of elution time versus drop time. In FIA-pH-DSTD, a commercial pH probe is used to measure pH during elution time, enabling surface pressure throughout drop time to be subsequently plotted as a function of eluting pH. An automated DSTD calibration procedure and data analysis method is applied, which allows simultaneous use of two different solvents, permitting real-time dynamic surface tension data to be obtained. The method was applied to the analysis of 14 commercial purified proteins, yielding characteristic features of surface activity as a function of pH. The reproducibility of the measurement and selectivity advantage of the DSTD was shown for the analysis of serum albumins from various mammalian sources. Several applications were also suggested and discussed in order to show the potential of the method for protein and food chemistry studies and in the study of protein-polymer interactions.

Concentration dependence of adsorption from human whole resting saliva at solid/liquid interfaces: An ellipsometric study

The rates and plateau values of adsorption at the solid/ liquid interface from human whole resting saliva from three male donors were studied in vitro. The influence of saliva concentration, the total protein content and the wettability of the exposed solids was investigated. Ellipsometry was used to study the rate and plateau values of the adsorption process in situ. The solid surfaces used were hydrophilic, negatively charged, silica and hydrophobic methylated silica. Salivary films were formed by adsorption from solutions of 0.05%, 0.10%, 0.20%, 0.50%, 1.0% and 2.0% (v/v) saliva in phosphate buffered saline. The results revealed that adsorption onto both types of surfaces was a rapid and, as expected, a concentration dependent process. The adsorbed amounts were in the range of monomolecular layers and were found to be larger onto the hydrophobic than the hydrophilic surfaces (p≤0.0062). A strong concentration dependence was denoted in all cases (p< 0.0001). Inter‐individual differences were found not to be significant (0.0636 ≤ p≤ 0.8508) at any of the saliva concentrations. However, an interaction was shown (p < 0.0001) between the protein concentrations and the donors, indicating qualitative differences in protein content and surface activity.