Effect of Ionization Degree of Poly(amidoamine) Dendrimer and 5-Fluorouracil on the Efficiency of Complex Formation-A Theoretical and Experimental Approach.

Due to their unique structure, poly(amidoamine) (PAMAM) dendrimers can bind active ingredients in two ways: inside the structure or on their surface. The location of drug molecules significantly impacts the kinetics of active substance release and the mechanism of internalization into the cell. This study focuses on the effect of the protonation degree of the G4PAMAM dendrimer and the anticancer drug 5-fluorouracil (5FU) on the efficiency of complex formation. The most favorable conditions for constructing the G4PAMAM-5FU complex are a low degree of protonation of the dendrimer molecule with the drug simultaneously present in a deprotonated form. The fluorine components in the XPS spectra confirm the formation of the stable complex. Through SAXS and DLS methods, a decrease in the dendrimer's molecular size resulting from protonation changes at alkaline conditions was demonstrated. The gradual closure of the dendrimer structure observed at high pH values makes it difficult for the 5FU molecules to migrate to the interior of the support structure, thereby promoting drug immobilization on the surface. The 1H NMR and DOSY spectra indicate that electrostatic interactions determine the complex formation process. Through MD simulations, the localization profile and the number of 5FU molecules forming the complex were visualized on an atomic scale.

The melting curves of calf thymus-DNA are buffer specific.

The specific effects of salts (strong electrolytes) on biomolecular properties have been investigated for more than a century. By contrast, the specific role of pH buffers (weak electrolytes and their salts) has usually been ignored. Here, specific buffer effects on DNA thermal stability were evaluated by measuring the melting curve of calf thymus DNA through UV-vis spectroscopy. The study was carried out using phosphate, Tris, citrate and cacodylate buffers at fixed pH 7.4 at concentrations varying systematically in the range 1-600 mM. DNA stability increases with buffer concentration and is influenced specifically by buffer type. To interpret empirical data, a theoretical model was applied with parameters quantifying the impact of buffer on the DNA backbone charge. Comparing the buffer effects via buffer ionic strength rather than buffer concentration, we find that the buffers stabilize DNA in the order Tris > cacodylate > phosphate > citrate.

BSA fragmentation specifically induced by added electrolytes: An electrospray ionization mass spectrometry investigation.

Biointerfaces are significantly affected by electrolytes according to the Hofmeister series. This work reports a systematic investigation on the effect of different metal chlorides, sodium and potassium bromides, iodides and thiocyanates, on the ESI/MS spectra of bovine serum albumin (BSA) in aqueous solution at pH = 2.7. The concentration of each salt was varied to maximize the quality of the ESI/MS spectrum, in terms of peak intensity and bell-shaped profile. The ESI/MS spectra of BSA in the absence and in the presence of salts showed a main protein pattern characterized by the expected mass of 66.5 kDa, except the case of BSA/RbCl (mass 65.3 kDa). In all systems we observed an additional pattern, characterized by at least three peaks with low intensity, whose deconvolution led to suggest the formation of a BSA fragment with a mass of 19.2 kDa. Only NaCl increased the intensity of the peaks of the main BSA pattern, while minimizing that of the fragment. NaCl addition seems to play a crucial role in stabilizing the BSA ionized interface against hydrolysis of peptide bonds, through different synergistic mechanisms. To quantify the observed specific electrolyte effects, two "Hofmeister" parameters (Hs and Ps) are proposed. They are obtained using the ratio of (BSA-Salt)/BSA peak intensities for both the BSA main pattern and for its fragment. SYNOPSIS: NaCl stabilizes BSA ion and almost prevents fragmentation due to denaturing pH.

Specific electrolyte effects on hemoglobin in denaturing medium investigated through electro spray ionization mass spectrometry.

We examine Hofmeister specific ion effects of electrolytes added to protein solution under conditions minimizing electrostatic attraction between cations and positively charged protein. Hemoglobin (Hb) in aqueous solution at the denaturing pH = 2.7 is investigated in the presence of several metal chlorides, along with sodium and potassium bromides, iodides and thiocyanates, using electrospray ionization mass spectrometry (ESI-MS). Salt concentration was varied to maximize peak intensity and bell-shaped profile in the ESI-MS spectrum. The α-chain of myoglobin is identified as the main pattern of the ESI-MS spectra in all Hb-salt systems. Both peak intensity and quality of the bell-shaped profile of the protein spectrum decrease in the cation order: K+ > > Mg2+ > Li+ > > Na+ > Ca2+ ≈ Cs+ > Rb+ for Hb-Metal Chloride systems, and decrease in the anion order: Cl- > Br- > I- > SCN- for systems of both Hb-NaX and Hb-KX salts. To quantify salt addition effects two Hofmeister specific electrolyte parameters HS, and PS are proposed. HS is the mean (Hb-salt)/Hb peak intensity ratio, measured for the nine peaks used for ESI-MS spectra deconvolution, taken at the same m/z values of the Hb profile. PS is the ratio between HS standard deviation and HS, and provides a specific perturbation parameter measuring the loss of protein structure. These two Hofmeister parameters give clear evidence of the effects induced either by KCl, MgCl2 and LiCl that enhance protein peak intensity, or by NaBr, NaI, NaSCN and KSCN that induce the protein fragmentation, due to electrolyte-mediated dissociation.

Aurivillius Oxides Nanosheets-Based Photocatalysts for Efficient Oxidation of Malachite Green Dye.

Aurivillius oxides ferroelectric layered materials are formed by bismuth oxide and pseu-do-perovskite layers. They have a good ionic conductivity, which is beneficial for various photo-catalyzed reactions. Here, we synthesized ultra-thin nanosheets of two different Aurivillius oxides, Bi2WO6 (BWO) and Bi2MoO6 (BMO), by using a hard-template process. All materials were characterized through XRD, TEM, FTIR, TGA/DSC, DLS/ELS, DRS, UV-Vis. Band gap material (Eg) and potential of the valence band (EVB) were calculated for BWO and BMO. In contrast to previous reports on the use of multi composite materials, a new procedure for photocatalytic efficient BMO nanosheets was developed. The procedure, with an additional step only, avoids the use of composite materials, improves crystal structure, and strongly reduces impurities. BWO and BMO were used as photocatalysts for the degradation of the water pollutant dye malachite green (MG). MG removal kinetics was fitted with Langmuir-Hinshelwood model obtaining a kinetic constant k = 7.81 × 10-2 min-1 for BWO and k = 9.27 × 10-2 min-1 for BMO. Photocatalytic dye degradation was highly effective, reaching 89% and 91% MG removal for BWO and BMO, respectively. A control experiment, carried out in the absence of light, allowed to quantify the contribution of adsorption to MG removal process. Adsorption contributed to MG removal by a 51% for BWO and only by a 19% for BMO, suggesting a different degradation mechanism for the two photocatalysts. The advanced MG degradation process due to BMO is likely caused by the high crystallinity of the material synthetized with the new procedure. Reuse tests demonstrated that both photocatalysts are highly active and stable reaching a MG removal up to 95% at the 10th reaction cycle. These results demonstrate that BMO nanosheets, synthesized with an easy additional step, achieved the best degradation performance, and can be successfully used for environmental remediation applications.

Buffer-specific effects arise from ionic dispersion forces.

Buffer solutions do not simply regulate pH, but also change the properties of protein molecules. The zeta potential of lysozyme varies significantly at the same buffer concentration, in the order Tris > phosphate > citrate, with citrate even inverting the zeta potential, usually positive at pH 7.15, to a negative value. This buffer-specific effect is a special case of the Hofmeister effect. Here we present a theoretical model of these buffer-specific effects using a Poisson-Boltzmann description of the buffer solution, modified to include dispersion forces of all ions interacting with the lysozyme surface. Dispersion coefficients are determined from quantum chemical polarizabilites calculated for each ion for tris, phosphate, and citrate buffer solutions. The lysozyme surface charge is controlled by charge regulation of carboxylate and amine sites of the component amino acids. The theoretical model satisfactorily reproduces experimental zeta potentials, including change of sign with citrate, when hydration of small cosmotropic ions (Na+, H+, OH-) is included.

Enzyme immobilization on metal organic frameworks: Laccase from Aspergillus sp. is better adapted to ZIF-zni rather than Fe-BTC.

Laccase from Aspergillus sp. (LC) was immobilized within Fe-BTC and ZIF-zni metal organic frameworks through a one-pot synthesis carried out under mild conditions (room temperature and aqueous solution). The Fe-BTC, ZIF-zni MOFs, and the LC@Fe-BTC, LC@ZIF-zni immobilized LC samples were characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The kinetic parameters (KM and Vmax) and the specific activity of the free and immobilized laccase were determined. Immobilized LCs resulted in a lower specific activity compared with that of the free LC (7.7 µmol min-1 mg-1). However, LC@ZIF-zni was almost 10 times more active than LC@Fe-BTC (1.32 µmol min-1 mg-1 vs 0.17 µmol min-1 mg-1) and only 5.8 times less active than free LC. The effect of enzyme loading showed that LC@Fe-BTC had an optimal loading of 45.2 mg g-1, at higher enzyme loadings the specific activity decreased. In contrast, the specific activity of LC@ZIF-zni increased linearly over the loading range investigated. The storage stability of LC@Fe-BTC was low with a significant decrease in activity after 5 days, while LC@ZIF retained up to 50% of its original activity after 30 days storage. The difference in activity and stability between LC@Fe-BTC and LC@ZIF-zni is likely due to release of Fe3+ and the low stability of Fe-BTC MOF. Together, these results indicate that ZIF-zni is a superior support for the immobilization of laccase.

Adsorption and Release of Sulfamethizole from Mesoporous Silica Nanoparticles Functionalised with Triethylenetetramine.

Mesoporous silica nanoparticles (MSN) were synthesised and functionalised with triethylenetetramine (MSN-TETA). The samples were fully characterised (transmission electron microscopy, small angle X-ray scattering, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential and nitrogen adsorption/desorption isotherms) and used as carriers for the adsorption of the antimicrobial drug sulphamethizole (SMZ). SMZ loading, quantified by UV-Vis spectroscopy, was higher on MSN-TETA (345.8 mg g-1) compared with bare MSN (215.4 mg g-1) even in the presence of a lower surface area (671 vs. 942 m2 g-1). The kinetics of SMZ adsorption on MSN and MSN-TETA followed a pseudo-second-order model. The adsorption isotherm is described better by a Langmuir model rather than a Temkin or Freundlich model. Release kinetics showed a burst release of SMZ from bare MSN samples (k1 = 136 h-1) in contrast to a slower release found with MSN-TETA (k1 = 3.04 h-1), suggesting attractive intermolecular interactions slow down SMZ release from MSN-TETA. In summary, the MSN surface area did not influence SMZ adsorption and release. On the contrary, the design of an effective drug delivery system must consider the intermolecular interactions between the adsorbent and the adsorbate.

Specific Buffer Effects on the Intermolecular Interactions among Protein Molecules at Physiological pH.

BSA and lysozyme molecular motion at pH 7.15 is buffer-specific. Adsorption of buffer ions on protein surfaces modulates the protein surface charge and thus protein-protein interactions. Interactions were estimated by means of the interaction parameter kD obtained from plots of diffusion coefficients at different protein concentrations (Dapp = D0[1 + kDCprotein]) via dynamic light scattering and nuclear magnetic resonance. The obtained results agree with recent findings confirming doubts regarding the validity of the Henderson-Hasselbalch equation, which has traditionally provided a basis for understanding pH buffers of primary importance in solution chemistry, electrochemistry, and biochemistry.

Specific Anion Effects on Lipase Adsorption and Enzymatic Synthesis of Biodiesel in Nonaqueous Media.

Pseudomonas fluorescens lipase (Pfl) was adsorbed on macroporous polypropylene to obtain a heterogeneous biocatalyst. The effect of NaCl concentration and of different 100 mm sodium salts on the Pfl loading and catalytic performance toward biodiesel synthesis via the solvent-free methanolysis of triglycerides was studied. Although lipase adsorption onto polypropylene is governed by hydrophobic interactions, both salt concentration and anion type affect lipase loading. Protein loading decreased along the series: Cl- > SO42- ≈ no salt > Br- > I- > SCN- > F- > AcO-. This nonmonotonic ion-specific trend can be the result of opposite mechanisms occurring during the adsorption step. A similar trend is observed also for triglyceride conversion and biodiesel yield. It is likely that ions affect the microenvironment of the adsorbed lipase by interacting specifically with the hydration water and polypeptide chains, thus affecting enzyme catalysis.

Specific ion effects on the enzymatic activity of alcohol dehydrogenase from Saccharomyces cerevisiae.

The enzymatic activity of alcohol dehydrogenase (ADH) in the presence of a range of electrolytes is investigated. In the presence of 150 and 200 mM cations a substantial increase in activity following the series GnCl < CsCl < KCl ∼ NaCl < LiCl was observed with a 69% increase in the presence of KCl 200 mM with respect to the salt-free solution. In the presence of 150 and 200 mM anions the increase in activity followed an ion specific trend NaF ∼ NaCl ∼ NaBr > no salt > NaClO4 > NaSCN with a peak in activity increase of 75% in the presence of NaBr. The values of the Michaelis-Menten constant (Km) did not show any significant ion specific effect, while the maximum rate (Vmax) of ethanol oxidation to acetaldehyde was strongly ion specific. The changes in specific activity and Vmax in the presence of anions likely arises from ion specific interactions with charged residues in the active site of ADH. The data indicate that the enzymatic activity of alcohol dehydrogenase can be modulated by the nature of electrolytes at physiological concentration.

Significant Enhancement of Structural Stability of the Hyperhalophilic ADH from Haloferax volcanii via Entrapment on Metal Organic Framework Support.

The use of an in situ immobilization procedure for the immobilization of hyperhalophilic alcohol dehydrogenase in a metal organic framework material is described. The easy and rapid in situ immobilization process enables retention of activity over a broad range of pH and temperature together with a decrease in the halophilicity of the enzyme. The catalytic activity of the immobilized enzyme was studied in nonaqueous solvent mixtures with the highest retention of activity in aqueous solutions of methanol and acetonitrile. The approach demonstrates that this immobilization method can be extended to hyperhalophilic enzymes with enhancements in activity and stability.

Specific cation effects on hemoglobin aggregation below and at physiological salt concentration.

Turbidity titrations are used to study the ion specific aggregation of hemoglobin (Hb) below and physiological salt concentration in the pH range 4.5-9.5. At a salt concentration 50 mM cations promote Hb aggregation according to the order Rb(+) > K(+) ~ Na(+) > Cs(+) > Li(+). The cation series changes if concentration is increased, becoming K(+) > Rb(+) > Na(+) > Li(+) > Cs(+) at 150 mM. We interpret the puzzling series by assuming that the kosmotropic Li(+) will bind to kosmotropic carboxylates groups-according to the law of matching water affinities (LMWA)-whereas the chaotropic Cs(+) will bind to uncharged protein patches due to its high polarizability. In fact, both mechanisms can be rationalized by invoking previously neglected ionic nonelectrostatic forces. This explains both adsorption to uncharged patches and the LMWA as a consequence of the simultaneous action of electrostatic and dispersion forces. The same interpretation applies to anions (with chaotropic anions binding to chaotropic amine groups). The implications extend beyond hemoglobin to other, still unexplained, ion specific effects in biological systems.