The Mobility of Chondroitin Sulfate in Articular and Artificial Cartilage Characterized by (13)C Magic-Angle Spinning NMR Spectroscopy

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in data:rat and artificial cartilage different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue. (C) 2010 Wiley Periodicals, Inc. Biopolymers 93: 520-532, 2010.

Spectrofluorimetric Determination of Second Critical Micellar Concentration of SDS and SDS/Brij 30 Systems

Potentially useful stead-state fluorimetric technique was used to determine the critical micellar concentrations (CMC(1) and CMC(2)) for two micellar media, one formed by SDS and the other by SDS/Brij 30. A comparative study based on conductimetric and surfacial tension measurements suggests that the CMC(1) estimated by the fluorimetric method is lower than the value estimated by these other techniques. Equivalent values were observed for SDS micelles without Brij 30 neutral co-surfactant. The use of acridine orange as fluorescent probe permitted to determine both CMC(1) and CMC(2). Based on it an explanation on aspects of micelle formation mechanism is presented, particularly based on a spherical and a rod like structures.

Sodium Tungstate on Some Biochemical Parameters of the Parotid Salivary Gland of Streptozotocin-Induced Diabetic Rats: A Short-Term Study

Several studies have shown the antidiabetic properties of sodium tungstate. In this study, we evaluated some biochemical parameters of the parotid salivary gland of streptozotocin-induced diabetic rats treated with sodium tungstate solution (2 mg/ml). The studied groups were: untreated control (UC), treated control (TC), untreated diabetic (UD), and treated diabetic (TD). After 2 and 6 weeks of treatment, parotid gland was removed and total protein and sialic acid (free and total) concentration and amylase and peroxidase activities were determined. Data were compared by variance analysis and Tukey test (p < 0.05). The sodium tungstate treatment modestly decreased the glycemia of streptozotocin-induced diabetic rats. At week 2 of the study, parotid gland of diabetic rats presented a reduction of total protein concentration (55%) and an increase of amylase (120%) and peroxidase (160%) activities, free (150%) and total (170%) sialic acid concentration. No alteration in the evaluated parameters at week 6 of the study was observed. Sodium tungstate presented no significant effect in parotid gland. Our results suggest that diabetes causes initial modification in biochemical composition of parotid. However, this gland showed a recovery capacity after 6 week of the experimental time. Sodium tungstate has no effect in peripheral tissues, such as salivary glands.

Conformational Properties of Angiotensin II and Its Active and Inactive TOAC-Labeled Analogs in the Presence of Micelles. Electron Paramagnetic Resonance, Fluorescence, and Circular Dichroism Studies

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents-negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)-was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC(1)-AII and inactive TOAC(3)-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more, pronounced for TOAC(3)-AII because of the proximity between the nitroxide and Tyr(4). CD spectra showed that although both AII and TOAC(1)-AII presented flexible conformations in water, TOAC(3)-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for All and TOAC(1)-AII, different conformations were acquired by TOAC(3)-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC(3)-AII is unable to acquire conformations similar to those of native AII and partially active TOAC(1)-AII is probably the explanation for its lack of biological activity. (C) 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 525-537, 2009.

Surface Activity of the Triflate Ion at the Air/Water Interface and Properties of N,N,N-Trimethyl-N-Dodecylammonium Triflate Aqueous Solutions

The surface activity of salts added to water is Air orders of magnitude lower than that of surfactants. Sodium trifluoromethanesulfonate (NaTf) produced a change in surface tension. with concentration, Delta gamma/Delta c, of -13.2 mN.L/m.mol. This value is ca. 4-fold larger than those of simple salts and that of methanesulfonate. This unexpected surface effect suggested that positively charged micelles containing Tf could exhibit interesting properties. Dodecyltrimethylammonium triflate (DTATf) had a higher Kraft temperature (37 degrees C) and a lower cmc (5 x 10(-3)M) and degree of dissociation (0.11) than the chloride and bromide salts of DTA. Above the Kraft temperature, at a characteristic temperature t(1), the addition of NaTf above 0.05 M. to a DTATf solution induced phase separation. By increasing the temperature of the two-phase system to above t(1), a homogeneous, transparent solution was obtained at a characteristic temperature t(2). These results, together with well-known triflate properties, led us to suggest that the Tf ion pairs With DTA and that the -CF(3) group may be dehydrated in the interfacial region, resulting in new and interesting self-aggregated structures.

Picosecond Dynamics of the Prototropic Reactions of 7-Hydroxyflavylium Photoacids Anchored at an Anionic Micellar Surface

Three water-insoluble, micelle-anchored flavylium salts, 7-hydroxy-3-octyl-flavylium chloride, 4`-hexyl-7-hydroxyflavylium chloride, and 6-hexyl-7-hydroxy-4-methyl-flavylium chloride, have been employed to probe excited-state prototropic reactions in micellar sodium dodecyl sulfate (SDS). In SDS micelles, the fluorescence decays of these three flavylium salts are tetraexponential functions in the pH range from 1.0 to 4.6 at temperatures from 293 to 318 K. The four components of the decays are assigned to Four kinetically coupled excited species in the micelle: specifically, promptly deprotonable (AH(+)*) and nonpromptly deprotonable (AH(h)(+)*) orientations of the acid in the micelle. the base-proton geminate pair (A*center dot center dot center dot H(+)), and the free conjugate base (A*). The initial prompt deprotonation to form the germinate pair occurs at essentially the same rate (k(d) similar to 6-7 x 10(10) s(-1)) for all three photoacids. Recombination of the germinate pair is similar to 3-fold faster than the rate of proton escape from the pair (k(rec) similar to 3 x 10(10) s(-1) and k(diss) similar to 1 x 10(10) s(-1)), corresponding to an intrinsic recombination efficiency of the pair of similar to 75%. Finally, the reprotonation of the short-lived free A* (200-350 ps, depending oil the photoacid) has two components, only one of which depends oil the proton concentration in the intermicellar aqueous phase. Ultrafast transfer of the proton to water and substantial compartmentalization of the photogenerated proton at the micelle surface Oil the picosecond time scale strongly suggest preferential transfer of the proton to preformed hydrogen-bonded water bridges between the photoacid and the anionic headgroups. This localizes the proton in the vicinity of the excited base much more efficiently than ill bulk water, resulting ill the predominance of geminate re reprotonation at the micelle surface.

Application of Microelectrode Voltammetry to Study the Properties of Surfactant Solutions: Alkyltrimethylammonium Bromides

Microelectrode cyclic voltammetry (MV) has been employed to investigate the micellar properties of solutions of homologous alkyltrimethylammonium bromides, RMe(3)ABr, R = C(10), C(12), and C(14), in water and in the presence of added NaBr. The micellar self-diffusion coefficient was calculated from the limiting current for the reversible electron transfer of micelle-bound ferrocene. From the values of this property, other parameters were calculated, including the micellar hydrodynamic radius, RH, and aggregation number, N(agg); the latter was also theoretically calculated. We determined the values of the diffusion coefficient as a function of various experimental variables and observed the following trends: The diffusion coefficient decreases as a function of increasing surfactant concentration (no additional electrolyte added); it decreases as a function of increasing surfactant concentration at fixed NaBr concentration; and it shows a complex dependence (increase then decrease) on the NaBr concentration at a fixed RMe(3)ABr concentration. The value of the intermicellar interaction parameter decreases and then increases as a function of increasing NaBr concentration. These results are discussed in terms of intermicellar,interactions and the effect of NaBr on the micellar surface charge density and sphere-to-rod geometry change. The NaBr concentration required to induce the latter change increases rapidly as a function of decreasing the length of R: no geometry change was detected for C(10)Me(3)ABr. Values of N(agg) increase as I function of increasing the length of R and are in good agreement with both literature values and values that were calculated theoretically. Thus, MV is a convenient and simple technique for obtaining fundamental properties of surfactant solutions, including additive-induced changes of micellar parameters (N(agg)) and morphology changes.

Effects of milk supplementation with skim milk powder, whey protein concentrate and sodium caseinate on acidification kinetics, rheological properties and structure of nonfat stirred yogurt

Milk supplementation with milk proteins in four different levels was used to investigate the effect on acidification and textural properties of yogurt. Commercial skim milk powder was diluted in distilled water, and the supplements were added to give different enriched-milk bases; these were heat treated at 90 degrees C for 5 min. These mixtures were incubated with the bacterial cultures for fermentation in a water bath, at 42 degrees C, until pH 4.50 was reached. Chemical changes during fermentation were followed by measuring the pH. Protein concentration measurements, microbial counts of Lactobacillus bulgaricus and Streptococcus thermophilus, and textural properties (G`, G ``, yield stress and firmness) were determined after 24 h of storage at 4 degrees C. Yogurt made with milk supplemented with sodium caseinate resulted in significant properties changes, which were decrease in fermentation time, and increase in yield stress, storage modulus, and firmness, with increases in supplement level. Microstructure also differed from that of yogurt produced with milk supplemented with skim milk powder or sodium caseinate. (C) 2009 Elsevier Ltd. All rights reserved.

The characterisation of the protective film formed by benzotriazole on the 90/10 copper-nickel alloy surface in H2SO4 media

The nature of the protective film formed by benzotriazole (BTAH) on the surface of the 90/10 CuNi alloy in deaerated 0.5 mol L-1 H2SO4 solution containing Fe(III) ions as oxidant was investigated by weight-loss, calorimetric measurements, and by surface-enhanced Raman spectroscopy (SERS). The SERS measurements show that the protective film is composed by the [Cu(I)BTA](n), polymeric complex and that the BTAH molecules are also adsorbed on the electrode surface. A modification of the BET isotherm for adsorption of gases ill solids is proposed to describe the experimental results obtained from weight-loss experiments that suggest an adsorption in multilayers. Electrochemical studies of copper and nickel in 0.5 mol L-1 H2SO4 in presence and absence of BTAH have also been made as an aid to interpret the results. The calculated adsorption free energy of the cuprous benzotriazolate on the surface of the alloy is in accordance with the value for pure copper. (C) 2007 Elsevier Ltd. All rights reserved.

Micellar properties of surface active ionic liquids: A comparison of 1-hexadecyl-3-methylimidazolium chloride with structurally related cationic surfactants

Ionic liquids, ILs, carrying long-chain alkyl groups are surface active, SAIIs. We investigated the micellar properties of the SAIL 1-hexadecyl-3-methylimidazolium chloride, C(16)MeImCl, and compared the data with 1-hexadecylpyridinium chloride, C(16)PYCl, and benzyl (3-hexadecanoylaminoethyl)dimethylammonium chloride, C(15)AEtBzMe(2)Cl. The properties compared include critical micelle concentration, cmc; thermodynamic parameters of micellization; empirical polarity and water concentrations in the interfacial regions. In the temperature range from 15 to 75 degrees C, the order of cmc in H(2)O and in D(2)O is C(16)PYCl > C(16)MeImCl > C(15)AEtBzMe(2)Cl. The enthalpies of micellization, Delta H(mic)(degrees), were calculated indirectly from by use of the van`t Hoff treatment; directly by isothermal titration calorimetry, ITC. Calculation of the degree of counter-ion dissociation, alpha(mic), from conductivity measurements, by use of Evans equation requires knowledge of the aggregation numbers, N(agg), at different temperatures. We have introduced a reliable method for carrying out this calculation, based on the volume and length of the monomer, and the dependence of N(agg) on temperature. The N(agg) calculated for C(16)PyCl and C(16)MeImCl were corroborated by light scattering measurements. Conductivity- and ITC-based Delta H(mic)(degrees) do not agree; reasons for this discrepancy are discussed. Micelle formation is entropy driven: at all studied temperatures for C(16)MeImCl; only up to 65 degrees C for C(16)PyCl; and up to 55 degrees C for C(15)AEtBzMe(2)Cl. All these data can be rationalized by considering hydrogen-bonding between the head-ions of the monomers in the micellar aggregate. The empirical polarities and concentrations of interfacial water were found to be independent of the nature of the head-group. (C) 2010 Elsevier Inc. All rights reserved.

Wettability of Aqueous Rhamnolipids Solutions Produced by Pseudomonas aeruginosa LBI

The wetting behavior of rhamnolipids produced by Pseudomonas aeruginosa LBI strain grown on waste oil substrate and sodium dodecyl sulfate (SDS) on glass, polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), poly(epsilon-caprolactone) (PCL) and polymer blend (PVC-PCL) was investigated by the measuring contact angle of sessile drops, to determine the wetting characteristics of rhamnolipids. The comparison of the wetting profiles showed that at low SDS and rhamnolipid concentrations, the contact angle increased and when the concentration of the surfactant increased further, the contact angle decreased. The blend surface (PVC-PCL) showed better wettability than the homopolymers themselves and the blend changed the surface hydrophobicity of the polymer, making it more hydrophilic. The rhamnolipids produced by the LBI strain exhibited superior wetting abilities than the chemical surfactant SDS one. This is the first work that evaluates the wetting properties of rhamnolipids on polymer blends.

On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations. HPS presented a smaller effect at concentrations above 1.5 mM. At 10 mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue. (C) 2011 Elsevier B.V. All rights reserved.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS): Effects of oligomeric dissociation

The present work focuses on the interaction between the zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp). Electronic optical absorption, fluorescence emission and circular dichroism spectroscopy techniques, together with Gel-filtration chromatography, were used in order to evaluate the oligomeric dissociation as well as the autoxidation of HbGp as a function of the interaction with HPS. A peculiar behavior was observed for the HPS-HbGp interaction: a complex ferric species formation equilibrium was promoted, as a consequence of the autoxidation and oligomeric dissociation processes. At pH 7.0, HPS is more effective up to 1 mM while at pH 9.0 the surfactant effect is more intense above 1 mM. Furthermore, the interaction of HPS with HbGp was clearly less intense than the interaction of this hemoglobin with cationic (CTAC) and anionic (SDS) surfactants. Probably, this lower interaction with HPS is due to two factors: (i) the lower electrostatic attraction between the HPS surfactant and the protein surface ionic sites when compared to the electrostatic interaction between HbGp and cationic and anionic surfactants, and (ii) the low cmc of HPS, which probably reduces the interaction of the surfactant in the monomeric form with the protein. The present work emphasizes the importance of the electrostatic contribution in the interaction between ionic surfactants and HbGp. Furthermore, in the whole HPS concentration range used in this study, no folding and autoxidation decrease induced by this surfactant were observed. This is quite different from the literature data on the interaction between surfactants and tetrameric hemoglobins, that supports the occurrence of this behavior for the intracellular hemoglobins at low surfactant concentration range. Spectroscopic data are discussed and compared with the literature in order to improve the understanding of hemoglobin-surfactant interaction as well as the acid isoelectric point (pI) influence of the giant extracellular hemoglobins on their structure-activity relationship. (c) 2007 Elsevier B.V. All rights reserved.

Interaction of bovine serum albumin (BSA) with ionic surfactants evaluated by electron paramagnetic resonance (EPR) spectroscopy

EPR spectra of 5- and 16-doxyl stearic acid nitroxide probes (5-DSA and 16-DSA, respectively) bound to bovine serum albumin (BSA) revealed that in the presence of ionic surfactants, at least, two label populations coexist in equilibrium. The rotational correlation times (tau) indicated that component I displays a more restricted mobility state, associated to the spin labels bound to the protein; the less immobilized component 2 is due to label localization in the surfactant aggregates. For both probes, the increase of surfactant concentration leads to higher motional levels of component 1 followed by a simultaneous decrease of this fraction of nitroxides and its conversion into component 2. For 10 mM cethyltrimethylammonium chloride (CTAC), the nitroxides are 100% bound to the protein, whereas at 10mM N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and sodium dodecyl sulfate (SDS) the fractions of bound nitroxides are reduced to 18% and 86%, respectively. No significant polarity changes were observed in the whole surfactant concentration range for component 1. Moreover, at higher surfactant concentration, component 2 exhibited a similar polarity as in the pure surfactant micelles. For 16-DSA the surfactant effect is different: at 10mM of HPS and CTAC the fractions of bound nitroxides are 76% and 49%, respectively, while at 10 mM SDS they are present exclusively in a micellar environment, consistent with 100% of component 2. Overall, both SDS and HPS are able to effectively displace the nitroxide probes from the protein binding sites. while CTAC seems to affect the nitroxide binding to a significantly smaller extent. (C) 2008 Elsevier B.V. All rights reserved.

Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 M Da, It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole range. Our zeta-potential data are consistent with light scattering results. Average values apt obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5,0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.