José Campos-Terán

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

Surface Adsorption and Bulk Aggregation of Cyclodextrins by Computational Molecular Dynamics Simulations as a Function of Temperature: α-CD vs β-CD

The structural simplicity of native cyclodextrins (CDs) contrasts with their complex behavior in the bulk of aqueous solutions, mainly when they are combined with other cosolutes. Many scientific and industrial applications based on these molecules are supported only by empirical information. The lack of fundamental knowledge, which would allow one to rationally optimize many of these applications, is notable mainly at the solution/air interface. Basic information on phenomena such as the spontaneous adsorption of native CDs or on the structure of CD aggregates in the bulk solution is really scarce. In order to fill these gaps, a detailed computational study on the adsorption and aggregation of α- and β-CDs as a function of temperature is presented here. Our simulations reproduce, at atomic resolution, the experimentally observed much higher ability of β-CD to aggregate compared to that of α-CD at 298 K, as well as their dependence on temperature. The adsorption of both individual CDs and small CD aggregates (up to 20 molecules) to the solution/air interface is found to be negligible. 0.8 μs long trajectories of single CD molecules in aqueous solution reveal that the main differences in the behavior of both CDs are their flexibility, higher for β-CD, and the occupancy of individual intramolecular hydrogen bonds that is significantly longer for the same cyclodextrin. The aggregation pattern of α- and β-CDs is followed at the hundreds of ns time scale, allowing both the spontaneous self-assembly of cyclodextrins and their redistribution along the aggregates to be observed. This is the first attempt to study the adsorption and aggregation of native cyclodextrins by atomistic molecular dynamics simulations.

Forces between Hydrophilic Surfaces Adsorbed with Apolipoprotein AII Alpha Helices

To provide better understanding of how a protein secondary structure affects protein-protein and protein-surface interactions, forces between amphiphilic alpha-helical proteins (human apolipoprotein AII) adsorbed on a hydrophilic surface (mica) were measured using an interferometric surface force apparatus (SFA). Forces between surfaces with adsorbed layers of this protein are mainly composed of electrostatic double layer forces at large surface distances and of steric repulsive forces at small distances. We suggest that the amphiphilicity of the alpha-helix structure facilitates the formation of protein multilayers next to the mica surfaces. We found that protein-surface interaction is stronger than protein-protein interaction, probably due to the high negative charge density of the mica surface and the high positive charge of the protein at our experimental conditions. Ellipsometry was used to follow the adsorption kinetics of this protein on hydrophilic silica, and we observed that the adsorption rate is not only controlled by diffusion, but rather by the protein-surface interaction. Our results for dimeric apolipoprotein AII are similar to those we have reported for the monomeric apolipoprotein CI, which has a similar secondary structure but a different peptide sequence and net charge. Therefore, the observed force curves seem to be a consequence of the particular features of the amphiphilic alpha-helices.

Complex Behavior of Aqueous α-Cyclodextrin Solutions. Interfacial Morphologies Resulting from Bulk Aggregation.

The spontaneous aggregation of α-cyclodextrin (α-CD) molecules in the bulk aqueous solution and the interactions of the resulting aggregates at the liquid/air interface have been studied at 283 K using a battery of techniques: transmission electron microscopy, dynamic light scattering, dynamic surface tensiometry, Brewster angle microscopy, neutron reflectometry, and ellipsometry. We show that α-CD molecules spontaneously form aggregates in the bulk that grow in size with time. These aggregates adsorb to the liquid/air interface with their size in the bulk determining the adsorption rate. The material that reaches the interface coalesces laterally to form two-dimensional domains on the micrometer scale with a layer thickness on the nanometer scale. These processes are affected by the ages of both the bulk and the interface. The interfacial layer formed is not in fast dynamic equilibrium with the subphase as the resulting morphology is locked in a kinetically trapped state. These results reveal a surprising complexity of the parallel physical processes taking place in the bulk and at the interface of what might have seemed initially like a simple system.

Immobilization effects on the photocatalytic activity of CdS quantum Dots-Horseradish peroxidase hybrid nanomaterials

The potential use of hybrid nanomaterials based on inorganic optically active nanoparticles known as quantum dots (QDs) and horseradish peroxidase (HRP) has been proposed by several authors as light-controllable nanocatalyzers, moreover, the immobilization within or over silica based supports represents an advantage over bulk-dispersed systems. However, the implications of the immobilization of such hybrid photoactivatable catalyzing systems have not been clarified with detail. Here, we present a thorough study of the functional photoactive efficiency and recycling of immobilized CdS QDs and HRP systems with different configurations, immobilized over silanized silica quartz crystal microbalance (QCM) sensors, allowing an accurate measure of the immobilized mass of each component and its correlation with the initial reaction rate of conversion of Amplex Red (AR) to resorufin. As well, the conversion efficiency is compared between the different systems and also to non-immobilized QD-HRP complexed systems.

Polymerization of 10,16-Dihydroxyhexadecanoic Acid, Main Monomer of Tomato Cuticle, Using the Lewis Acidic Ionic Liquid Choline Chloride·2ZnCl2

10,16-dihydroxyhexadecanoic acid, main monomer of the tomato cuticle obtained from agro-residual wastes, was polymerized using (choline chloride.2ZnCl2) ionic liquid as catalyst at three different temperatures (80, 90 and 100 °C). The resulting polyesters obtained under these conditions were insoluble in most of the organic solvents and showed different physicochemical properties. While at 80 °C polymers were obtained as powder, at higher temperature they were found in viscous consistency. According with the CP MAS 13C NMR and FTIR-ATR analysis, polymers showed a linear structure with an increasing degree of esterification in position C-10. Polyesters were analyzed by means of differential scanning calorimetry (DSC), atomic force microscopy (AFM) and X ray diffraction (small- and wide-angle scattering, SWAXS) techniques.

Biosensors based on oxidative enzymes for detection of environmental pollutants

Abstract In recent years, the continuous and accumulative discharge of toxic and contaminating compounds to the environment makes necessary to propose precise and quick methods for their detection and quantitation. Especially when one considers that the environmental impact of some of these emerging contaminants has not been clearly determined. Enzyme-based biosensors are an interesting alternative when inspecting different pollutants present in the environment in a quick, efficient, automatized, and economic way. Oxidative enzymes such as peroxidases and polyphenol oxidases (laccases and tyrosinases) are versatile and highly functional enzymes used for analyte recognition. Therefore, these enzymes are considered attractive and interesting biomolecules to act as recognition elements in biosensors. In this regard, detection of pollutants such as pesticides, phenols, heavy metals, and pharmaceutical compounds by using oxidative enzymes as recognition elements in biosensors is a versatile field, and it is the focus of the present review.

Free-lignin cellulose obtained from agar industry residues using a continuous and minimal solvent reaction/extraction methodology

Herein we present a green procedure to obtain cellulose (Cel) polymers with different physicochemical properties at high purity, and underline its two major aspects: sustainability and efficiency. In the first place, regarding sustainability, the source of Cel was residues from agar industries, which are based on red seaweed and hence free of lignin, thus facilitating the extraction of Cel. In the aspect of efficiency, a continuous extraction/reaction system was used to obtain pure Cel from these residues. The extraction/reaction device used in this study normally works in a liquid–liquid extraction fashion, but in this particular case it was successfully employed as a liquid–solid system. This methodology is important, because it concomitantly reduces the time of extraction/reaction procedures in the same flask and also minimizes the amount of solvent used. Thus high purity Cel was obtained using a continuous and minimal solvent extraction/reaction system in neutral/acidic/basic conditions leading to Celn/Cela/Celb polymers in 42/34/43.3% yield. These materials were characterized by 13C cross-polarization magic-angle spinning (CP-MAS) NMR, Fourier transform infrared spectroscopy (FT-IR), CHNS elemental analyses, X-ray diffraction (XRD), size exclusion chromatography (SEC) and compared against microcrystalline cellulose (MCC), confirming chemical integrity. Crystallinity index (CI [%]), was obtained from XRD/CP-MAS NMR data. All samples had slightly higher crystallinity than that of MCC. Molecular weight (MW, g mol−1), polydispersity index (PDI) and degree of polymerization (DP) for Celn, Cela, Celb polymers were all higher than those in MCC. Compared to MCC, the physicochemical characteristics of the isolated Cel polymers varied depending on the treatment, neutral being the mildest. The greener procedures developed herein provide Cel suitable for research and development of Cel derived substances.

Conformational and Disorder to Order Transitions in Proteins: Structure / Function Correlation in Apolipoproteins

The concept of protein folding is directly related with the process of reversible disorder-toorder transitions, by which an unfolded polypeptide chain folds into a specific functional native structure (Eaton et al., 2000; Rose et al., 2006). For folding into a native state, unfolded polypeptide chains require the intervention of weak interactions. Driven by hydrophobic interactions, a polypeptide chain begins to fold when placed in an aqueous medium, and rapidly becomes a molten globule followed by an important release of latent heat. Stabilization of the molten globule is achieved mainly through the distribution of hydrophobic residues away from the water matrix. On the other hand, because the polar residues contained in a protein develop hydrogen bonds with the water network as well as with each other, α-helices and β-sheets can be formed when bonds switch between molecules. It has been calculated that such bonds might be in the order of 10-12 s, very similar to those we find in water itself. The random equilibrium can be shifted toward one of these conformations by means of two stages: a fast stage, during which the unfolded polypeptide becomes a molten globule; and a slow stage, in which the molten globule slowly transforms into a fully folded form or native state (Huang, 2005). These two stages in protein folding can be illustrated by a ‘‘folding funnel’’, during which due to a small change in entropy with a large loss of energy, a molten globule evolves into the native state (Fig. 1a) (Dobson, 2003; Gsponer & Vendruscolo, 2006).

The cis-bis(decanoate)tin phthalocyanine/DPPC film at the air/water interface.

Films made of cis-bis-decanoate-tin(IV) phthalocyanine (PcSn10) and racemic dipalmitoylphosphatidylcholine (DPPC) are studied with compression isotherms and Brewster angle microscopy (BAM) at the air/water interface. Films enriched in PcSn10 present phase separation elliptical-shaped domains. These domains present optical anisotropy and molecular order. They are enriched in PcSn10, and the film outside these domains is enriched in DPPC, as shown in by high-angle annular dark-field transmission electron microscopy on Langmuir-Blodgett (LB) transferred films. Film collapse area and atomic force microscopy images of LB transferred films on mica indicate that the films are actually multilayers. A computational survey was performed to determine how the PcSn10 molecules prefer to self-assemble, in films basically made of PcSn10. The relative energetic stability for several dimeric assemblies was obtained, and a crystal model of the film was developed through packing and repeating the PcSn10 molecules, along the crystallographic directions of the unit cell. Our results contribute to understanding the strong interaction between PcSn10 and DPPC at the air/water interface, where even small quantities of DPPC (~1-2%) can modify the film in an important way.