Inorganic anions in goat and ovine milk from Calabria (Italy) by suppressed ion chromatography.

Forty-seven samples of milk taken from goat and ovine farms in Calabria (southern Italy) between April and May 2010 were analysed. Analyses were carried out by ion chromatography (ICS 1000; Dionex). For method validation, RSDs (%) on retention times and on peak area were determined by considering a mixture of standard anions at the concentration level of 0.1-30 mg l(-1) for chloride, bromide, nitrite, nitrate, sulfate and phosphate. The higher RSDs were 1.4% and 2.2% for retention times and 2.4% and 3.7% for areas for intra- and inter-day repeatability, respectively. Good linearity was observed in each concentration range, with linear correlation coefficients (R(2)) better than 0.987. The LODs and LOQs were calculated experimentally as signal-to-noise ratios of 3 and 10, respectively. Analysis of the results obtained indicated that phosphates were the most abundant anion (1409-3050 mg l(-1)) followed by chloride (679-2568 mg l(-1)) and sulfates (25-341 mg l(-1)). Nitrates were found to be lower at 146 mg l(-1) (the maximum value observed). The values of anions found in this work are in agreement with those reported in the literature for cow's milk. There are no significant differences between ovine and goat's milk samples. The method used for this analysis of anions combines rapidity with high precision, accuracy and sensitivity; therefore, it is suitable for routine analysis. Moreover, one of the most obvious advantages of this technique is that multiple elements can be determined in one sample with no serial dilutions and the complete analysis can be performed by using only one instrument.

Amyloid fibrils formation of concanavalin A at basic pH.

Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular ß-sheet structure which constitutes a rate-limiting step of the process. The intertwining between the formation of ß-aggregate structures and the whole aggregation process is discussed as a function of protein concentration: a coagulation process produces the same kind of aggregates at the different concentrations studied. Multiangle light-scattering data highlight the onset of the condensation process which gives rise to formation of compact fractal aggregates. AFM microscopy supports this conclusion showing thin fibrils of ConA, formed in the early stage of aggregation, which further interact to form larger structures with a netlike spatial organization.

Quantification of underivatized fatty acids from vegetable oils by HPLC with UV detection.

We propose a chromatographic method for the separation of saturated and unsaturated fatty acids by a high-performance liquid chromatography system, equipped with a photo diode array detector. Central to the method is the use of an appropriate mobile phase composed of acetonitrile, methanol, and n-hexane in ratio 90:8:2 acidified with 0.2% acetic acid, which allows the detection of fatty acids without a preliminary derivatization with chromophores or fluorescent dyes. Calibration on solutions of standards mixtures gives a quantification limit (at a wavelength of 208 nm) of 0.232, 0.093, 0.039, 0.056, 0.068, 0.004, 0.0005, 0.067 mg/mL for the myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and erucic acids, respectively. The method, applied to different vegetable oils (olive, sunflower, soybean, and palm) was able to distinguish the main fatty acids and quantify their amount. Data reliability was tested by comparing our results (on the relative percentages of some fatty acids in the olive oil) with those obtained by gas chromatographic analysis. Differences of the order of 0.3%, 0.6%, 2%, and 6% were observed for the oleic, linoleic, palmitic, and linolenic acids. Although less accurate, our method proved to be a simple alternative to standard gas chromatographic technique, as it can be applied even using a simple UV detector.

Relation between structural and release properties in a polysaccharide gel system.

The potential utility of kappa-carrageenan gels for preparing drug release devices is here shown. Structural properties of kappa-carrageenan gels prepared with different salt composition and containing Ketoprofen sodium salt, as model drug, have been evaluated with static light scattering and rheological measurements. These properties have been correlated with release profiles in vitro at pH 5.5. Release properties from gelled matrices have been compared with those obtained by two commercial products containing the same drug. Results show that: i) in this system it is possible to easily control the gel texture by using different cationic concentration; ii) the kinetics of drug release by kappa-carrageenan gels are dependent on the structural properties of matrices; iii) in the typical interval time used in classical local applications, all gel samples release the loaded drug almost completely, at difference with the commercial products. All these findings can provide useful suggestions for the realization of classical topical release systems.

Role of charges and solvent on the conformational properties of poly(galacturonic acid) chains: a molecular dynamics study.

Pectin shares with many other polysaccharides an intrinsic chemical and physical complexity. The widespread industrial applications have made it one of the most studied polysaccharides. This work presents a theoretical model of poly(galacturonic acid), the major constituent of pectin, suitable to study its structural and dynamical properties. In particular, the effects of solvent and charge status are studied. The dynamics is shown to be severely affected by the presence of charged groups on each residue, making the charged chain much more rigid than the uncharged one. A key structural property for a semirigid polymer, the asymptotic persistence length, is calculated for relatively short charged and uncharged chains in molecular water solvent using a new method. The influence of charge on structural properties of poly(galacturonic acid) is shown to be strong and solvent-dependent. In fact, a large difference is found between continuum solvent adiabatic map calculations and molecular dynamics with explicit solvent, with the latter showing a much larger persistence length.

K(+) and Na(+) effects on the gelation properties of kappa-Carrageenan.

The effects of K(+), Na(+) ions and their mixture on the conformational transition and macroscopic gel properties of kappa-Carrageenan system have been studied using different experimental techniques. The macroscopic gelation properties of kappa-Carrageenan were found to be dependent upon cosolute type. Indeed, a more ordered and strong gel was obtained in the presence of K(+) with respect to Na(+) ions. The gel properties obtained using mixtures of two cosolutes are shown to depend on the [K(+)]/[Na(+)] ratio.

Thermoreversible gelation of kappa-carrageenan: relation between conformational transition and aggregation.

We have studied, by optical rotation dispersion, light scattering and rheology, the kappa-Carrageenan system to elucidate the processes involved in gel formation (on decreasing the temperature) and gel melting (on increasing the temperature). Our results show that, on decreasing the temperature, a conformational transition from coils to double helices first occurs, followed by aggregation of the double helices into domains and gel formation at appropriate polymer concentration. Structural details of this sequence are better revealed by re-heating the system. Melting appears as a two-step process characterized by first a conformational change of helices involved in junction zones between aggregates, followed by the conformational transition of the helices inside the aggregates. These helices can regain the coil conformation only when the aggregates melt at higher temperature, in full agreement with the old 'domain' model. The full description of the sol-gel mechanism of this system can be useful in the search for new methods to control the gel texture, a relevant property for many industrial applications.

Effects of intermediates on aggregation of native bovine serum albumin.

Protein aggregation has been recognized to be a pathological indicator for several fatal diseases, such as Alzheimer's disease, transmissible spongiform encephalopathies, Creutzfeldt-Jacob disease, etc. Aggregation usually involves conformational changes of proteins that have acquired an intermediate beta-structure-rich conformation and can occur even at low protein concentration. Recent work in our laboratory has shown that bovine serum albumin (BSA), even at low-concentration, exhibits self-association properties related to conformational changes, so providing a very convenient model system to study this class of problems. Here we report data (obtained by different experimental techniques) on a mixture of BSA in native and intermediate (beta-structure-rich) form. Results show that the interaction between the two species is responsible for a decrease in the thermodynamic stability of the solution. This occurs without requiring noticeable conformational changes of the native protein. Results presented here can provide new insight on the "protein only" hypothesis proposed for the formation of plaques involved in several neurodegenerative diseases.

Interaction of processes on different length scales in a bioelastomer capable of performing energy conversion.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions. Previous studies have shown that aggregation is prompted by the (diverging) critical fluctuations of concentration occurring in the solution, in vicinity of its spinodal line. Here, we present combined circular dicroism (CD) and light scattering experiments, and independent fittings of experimental data to the theoretical spinodal and binodal (coexistence) lines. Results show the following logical and causal sequence of processes: (a) Smooth and progressive conformational changes promoted by concentration fluctuations occurring as temperature is raised "pull down" (in the temperature scale) the instability region of the solution. (b) This further promotes critical fluctuations. (c) The related locally high concentration prompts a further substantial conformational change ending in triple-helix formation and coacervation. (d) This intertwining of processes, covering different length scales (from that of individual peptides to the mesoscopic one of demixed regions), is related to the fact that solvent-induced interactions play a strong role over the entire scale span. These results concur with other recent ones in pointing out that process interactions over many length-scales probably reflect a frequent if not ubiquitous pattern in protein aggregation. This may be highly relevant to the desirable deep understanding of such phenomenon, whose interests cover many fields.

Effects of electric charges on hydrophobic forces. II

We study by molecular-dynamics simulations the effect of electric charges of either sign on hydrophobic interactions and on the dynamics of hydration water, using explicit water and very simplified solutes. Results show that the presence of a charged solute can disrupt the "hydrophobic contact bond" between two apolar solutes nearby, by forcing them towards a different configuration. As a consequence of different structural changes of the solvent caused by charges of opposite sign, the effect is markedly charge-sign-dependent. Analogous weaker effects appear to be induced by the presence of one additional apolar element. The dynamics of hydration water around each solute is also seen to be strongly influenced by the presence of other (charged or uncharged) nearby solutes. Comparison between our results on hydration water dynamics around charged solutes and available experimental data allows sorting out the effects of solute charge sign and size. Our results also offer a plain interpretation of the equivalence of the effects on water structure due to solute ions and to high pressures. These results reflect at a basic paradigmatic level the immensely more complex cases of well-known phenomena such as salting-in and salting-out, and of protein conformational changes caused, e.g., by the arrival of a charged or of an apolar group (phosphorilation or methylation). As it will be discussed, they help in the direction of Delbruck's desirable "progress towards a radical physical explanation" for this class of phenomena.

Interacting processes in protein coagulation.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute.

Molecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the system and hydration reconfiguration is also elicited. Switching is seen to occur on a time scale two orders of magnitude longer than that of the reconfiguration time of the solute taken alone, or that of the unperturbed solvent. Qualitatively, these results are unaffected by a different choice of the water-water interaction potential. They show that already at an elementary level, solvent-induced interactions alone, when fully accounted for, can be responsible for configurational and dynamical features essential to protein folding and function.

Interaction of explicit solvent with hydrophobic/philic/charged residues of a protein: residue character vs. conformational context.

Molecular dynamics simulations of model solutes in explicit molecular water have recently elicited novel aspects of the strong nonpair additivity of the potential of mean force (PMF) and related solvent-induced forces (SIFs) and hydration. Here we present the results of the same type of work on SIFs acting on bovine pancreatic trypsin inhibitor (BPTI) at single residue/sidechain resolution. In this system, nonpair additivity and the consequent dependence of SIFs on the protein conformational context are sufficiently strong to overturn SIFs on some individual residues, relative to expectations based on their individual characters. This finding calls for a revisitation and offers a richer and diversified understanding of the role of hydrophobic/philic/charged groups in establishing the exquisite specificity of biomolecular folding and functional conformation. Its relevance is appreciated by noting that the work of a typical SIF acting on one residue, when displaced across a distance of 1 A, is the equivalent of up to a few kcal/mol, which is the range of the stability/function free energy of a protein.

Collective properties of hydration: long range and specificity of hydrophobic interactions.

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement recent data concerning SIFs on a protein at single-residue resolution and on model systems. They illustrate new important consequences of the collective character of hydration and of PMF and reveal new aspects of hydrophobic interactions and, in general, of SIFs. Their relevance to protein recognition, conformation, function, and folding and to the observed slight yet significant nonadditivity of functional effects of distant point mutations in proteins is discussed. These results point out the functional role of the configurational and dynamical states (and related statistical weights) corresponding to the complex configurational energy landscape of the two interacting systems: biomolecule + water.