Separation of proteins by hydrophobic interaction chromatography at low salt concentration

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.     

Specific salt effects in hydrophobic interaction chromatography of proteins

Summary It has been noted in the literature that certain salts enter into specific interaction with proteins. As a result of this, they may act as salting-in agents. We have investigated the effect of magnesium chloride which is known to possess such unusual properties on the retention of proteins in hydrophobic-interaction chromatography. First the retention behaviour of amino acids and small peptides having a wide polarity range was studied on reversed-phase columns using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂, in wide the concentration ranges. For less polar eluites plots of the logarithmic retention factors against the salt concentration were found to be linear, whereas the more polar species showed irregular behavior. The retention of a wide range of proteins was measured on a TSK Phenyl-5-PW column using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂ at different concentrations.The salt-mediated retention was regular with (NH₄)₂ SO₄ and MgSO₄ although MgSO₄ showed a lesser effect than that predicted by the surface-tension increment. The effect of MgCl₂ was quite irregular: the retention factors either increased or decreased or remained unchanged depending on the protein. These results corroborate earlier observations regarding the particular effect of MgCl₂ and suggest the modulation of selectivity in hyrophobic-interaction chromatography by the addition of MgCl₂ to the eluent.     

Development of novel statistical potentials describing cation-pi interactions in proteins and comparison with semiempirical and quantum chemistry approaches

Novel statistical potentials derived from known protein structures are presented. They are designed to describe cation-pi and amino-pi interactions between a positively charged amino acid or an amino acid carrying a partially charged amino group and an aromatic moiety. These potentials are based on the propensity of residue types to be separated by a certain spatial distance or to have a given relative orientation. Several such potentials, describing different kinds of correlations between residue types, distances, and orientations, are derived and combined in a way that maximizes their information content and minimizes their redundancy. To test the ability of these potentials to describe cation-pi and amino-pi systems, we compare their energies with those computed with the CHARMM molecular mechanics force field and with quantum chemistry calculations at the Hartree-Fock level (HF) and at the second order of the M?ller-Plesset perturbation theory (MP2). The latter calculations are performed in the gas phase and in acetone, in order to mimic the average dielectric constant of protein environments. The energies computed with the best of our statistical potentials and with gas-phase HF or MP2 show correlation coefficients up to 0.96 when considering one side-chain degree of freedom in the statistical potentials and up to 0.94 when using a totally simplified model excluding all side-chain degrees of freedom. These potentials perform as well as, or better than, the CHARMM molecular mechanics force field that uses a much more detailed protein representation. The good performance of our cation-pi statistical potentials suggests their utility in protein structure and stability prediction and in protein design.     

Solute-solvent interactions probed by intermolecular NOEs

Nuclear Overhauser effects arising from the interactions of spins of solvent molecules with spins of a solute should reveal the "exposure" of solute spins to collisions with solvent. Such intermolecular NOEs could, therefore, provide information regarding conformation or structure of the solute. Determinations of solute-solvent NOEs of 1,3-di-tert-butylbenzene in solvents composed of perfluoro-tert-butyl alcohol, tetramethylsilane, and carbon tetrachloride have been carried out. A crude, but apparently reliable, method for prediction of intermolecular solvent-solute NOEs based on hard (noninteracting) spheres was developed. Comparison of experimental to predicted NOEs indicates that tetramethylsilane interacts with the solute according to the model. By contrast, intermolecular NOE data indicate attractive interactions between the solute and perfluoro-tert-butyl alcohol. All NOE results and the corresponding predictions confirm that proton H2 of the solute is protected by the flanking tert-butyl groups from interactions with solvent molecules.     

Effect of salt on the inter- and intramolecular hydrophobic interactions of macromolecules

The effect of salt on the structure of a low density lipoprotein (LDL) and on the reversible polymerization of bovine serum albumin (BSA) reduced with 2-mercaptoethanol was investigated by means of ultracentrifugal analysis. The chaotropic anion, e.g., SCN^– and I^–, at 5M completely disrupted the LDL structure and effectively dissociated BSA oligomers at lower concentrations. The parallelism between the anion order of these effects and that of the chaotropic effect suggested that the observed salt effects are primarily based on the disruption of hydrophobic interactions. The cation effectiveness disrupting the LDL structure followed the order of their promoting effect on the water structure, i.e., Li⁺>Na⁺>K⁺>Cs⁺. However, Cs⁺ was most effective in dissociating BSA oligomers, and this was attributed to the -complex formation with the aromatic amino acid side chains which otherwise contribute to the promotion of the intermolecular hydrophobic association.     

Stabilization of beta-hairpin peptides by salt bridges: role of preorganization in the energetic contribution of weak interactions

A model beta-hairpin peptide has been used to investigate the context-dependent contribution of cross-strand Lys-Glu interactions to hairpin stability. We have mutated two Ser-Lys interstrand pairs to Glu-Lys salt bridges, one close to the type I' Asn-Gly turn sequence (Ser6 --> Glu), and one close to the N- and C-termini (Ser15 --> Glu). Each individual interaction contributes approximately 1.2-1.3 kJ mol(-1) to stability; however, introducing the two salt bridges simultaneously produces a much larger overall contribution (-3.6 kJ mol(-1)) consistent with an important role for preorganization and cooperativity in determining the energetics of weak interactions. We compare and contrast CD and NMR data on the highly folded hairpin with the two Glu-Lys pairs to shed light on the nature of the folded state in water. We show that large cosolvent-induced changes in the CD spectrum, in contrast with the modest effects observed on Halpha chemical shifts, support a hydrophobically collapsed entropy-driven conformation in water whose stability is modulated by long-range Coulombic interactions from the Glu-Lys interactions. Cosolvent stabilizes the structure enthalpically, as is evident from CD melting profiles.     

Enthalpy-entropy contributions to salt and osmolyte effects on molecular-scale hydrophobic hydration and interactions

Salts and additives can significantly affect the strength of water-mediated interactions in solution. We present results from molecular dynamics simulations focused on the thermodynamics of hydrophobic hydration, association, and the folding-unfolding of a hydrophobic polymer in water and in aqueous solutions of NaCl and of an osmolyte trimethylamine oxide (TMAO). It is known that addition of NaCl makes the hydration of hydrophobic solutes unfavorable and, correspondingly, strengthens their association at the pair as well as the many-body level (Ghosh, T.; Kalra, A.; Garde, S. J. Phys. Chem. B 2005, 109, 642), whereas the osmolyte TMAO has an almost negligible effect on the hydrophobic hydration and association (Athawale, M. V.; Dordick, J. S.; Garde, S. Biophys. J. 2005, 89, 858). Whether these effects are enthalpic or entropic in origin is not fully known. Here we perform temperature-dependent simulations to resolve the free energy into entropy and enthalpy contributions. We find that in TMAO solutions, there is an almost precise entropy-enthalpy compensation leading to the negligible effect of TMAO on hydrophobic phenomena. In contrast, in NaCl solutions, changes in enthalpy dominate, making the salt-induced strengthening of hydrophobic interactions enthalpic in origin. The resolution of total enthalpy into solute-solvent and solvent-solvent terms further shows that enthalpy changes originate primarily from solvent-solvent energy terms. Our results are consistent with experimental data on the hydration of small hydrophobic solutes by Ben-Naim and Yaacobi (Ben-Naim, A.; Yaacobi, M. J. Phys. Chem. 1974, 78, 170). In combination with recent work by Zangi, Hagen, and Berne (Zangi, R.; Hagen, M.; Berne, B. J. J. Am. Chem. Soc. 2007, 129, 4678) and the experimental data on surface tensions of salt solutions by Matubayasi et al. (Matubayasi, N.; Matsuo, H.; Yamamoto, K.; Yamaguchi, S.; Matuzawa, A. J. Colloid Interface Sci. 1999, 209, 398), our results highlight interesting length scale dependences of salt effects on hydrophobic phenomena. Although NaCl strengthens hydrophobic interactions at both small and large length scales, that effect is enthalpy-dominated at small length scales and entropy-dominated for large solutes and interfaces. Our results have implications for understanding of additive effects on water-mediated interactions, as well as on biocompatibility of osmolyte molecules in aqueous solutions.     

Mucin-electrolyte interactions at the solid-liquid interface probed by QCM-D

The interaction between mucin and ions has been investigated by employing the quartz crystal microbalance technique with measurement of energy dissipation. The study was partially aimed at understanding the adsorption of mucin on surfaces with different chemistry, and for this purpose, surfaces exposing COOH, OH, and CH(3) groups were prepared. Mucin adsorbed to all three types of functionalized gold surfaces. Adsorption to the hydrophobic surface and to the charged hydrophilic surface (COOH) occured with high affinity despite the fact that in the latter case both mucin and the surface were negatively charged. On the uncharged hydrophilic surface exposing OH groups, the adsorption of mucin was very low. Another aim was to elucidate conformational changes induced by electrolytes on mucin layers adsorbed on hydrophobic surfaces from 30 mM NaNO(3). To this end, we investigated the effect of three electrolytes with increasing cation valance: NaCl, CaCl(2) and LaCl(3). At low NaCl concentrations, the preadsorbed layer expands, whereas at higher concentrations of NaCl the layer becomes more compact. This swelling/compacting of the mucin layer is fully reversible for NaCl. When the mucin layer instead is exposed to CaCl(2) or LaCl(3), compaction is observed at 1 mM. For CaCl(2), this process is only partially reversible, and for LaCl(3), the changes are irreversible within the time frame of the experiment. Finally, mucin interaction with the DTAB cationic surfactant in an aqueous solution of different electrolytes was evaluated with turbidimetry measurements. It is concluded that the electrolytes used in this work screen the association between mucin and DTAB and that the effect increases with increasing cation valency.     

Simultaneous correlation of hydrophobic interactions in HIC and protein solubility in aqueous salt solutions and mixed solvents

The chromatographic retention in hydrophobic and reversed phase chromatography and the solubility of proteins display some common features. The chromatographic retention, as well as the solubility, is modulated by the thermodynamic properties of the solute in the fluid phase. The retention measurements at linear conditions provide information of the solution properties of the protein at infinite dilution, and the solubility measurements produce the supplementary information about the solution properties at the saturation limit. This provides a useful approach to simultaneous correlation of the chromatographic retention and the solubility.The experimental data, used for the correlation, comprise retention measurements of lysozyme on different HIC adsorbents using an aqueous ammonium sulphate eluant, an aqueous ammonium sulphate eluant with an admixture of ethanol, as well as published solubility data.The chromatographic retention data and the corresponding solubility data have been correlated using a chemical potential model derived from Kirkwood's theory of solutions of charged macro-ions and zwitterions in electrolyte solutions. The model correlated the chromatographic retention factor and the solubility data within the precision of the measurements. The model was applied in a pH range from 4 to 11. It was demonstrated experimentally, as well as theoretically, that an admixture of ethanol to the aqueous eluant changes the thermodynamic retention factor on various adsorbents identically when compared to the thermodynamic retention factor in an ethanol free eluant.     

Intermolecular interactions in electroactive thiol monolayers probed by linear scan voltammetry

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Charge transfer interactions in liquid crystalline polymers probed by fluorescence spectroscopy

Rigid-rod aromatic LC polyester with long alkyl side chains and two thermotropic LC polyesters (PET40/OBA60 and PB-10) were studied by fluorescence spectroscopy to investigate their charge transfer interactions corresponding to LC configuration and changes during phase transition.     

Solubilization of hydrophobic molecules in nanoparticles formed by polymer-surfactant interactions

The interaction between the anionic surfactant, sodium dodecyl sulfate, and the polyelectrolyte, poly(diallyldimethylammonium chloride), may lead to formation of nanoparticles dispersed in water. The morphology of the resulting nanoparticles and their ability to solubilize hydrophobic molecules were evaluated. As shown by SEM and AFM imaging, the particles are spherical, having a diameter of about 20 nm. The solubilization within the nanoparticles was tested with pyrene, a fluorescence probe, and Nile Red, a solvatochromic probe. It was found that for Nile Red the solubilization within the nanoparticles is at lower polarity than for SDS micelles, and from pyrene solubilization it appears that the hydrophobicity of the nanoparticles depends on the ratio between the SDS molecules and the charge unit of the polymer.     

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.     

Divalent metal ion-peptide interactions probed by electron capture dissociation of trications

Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP + H + M]3+ (M2+ = Mg2+ -Ba2+ and Mn2+ -Zn2+) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c' and z* -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y'-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (cn 'M - H)+ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful selection of the cationizing agent.     

Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.

Hydrogen-exchange electrospray-ionization mass spectrometry is demonstrated to be an effective new method for probing conformational changes of proteins in solutions. The method is based on the mass spectrometric measurement of the extent of hydrogen/deuterium exchange that occurs in different protein conformers over defined periods of time. Results are presented in which hydrogen-exchange electrospray-ionization mass spectrometry is used to probe conformational changes in bovine ubiquitin induced by the addition of methanol to aqueous acidic solutions of the protein.     

Thermal unfolding of proteins probed by laser spray mass spectrometry

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.     

Ion decomposition versus molecular size probed by vacuum ultraviolet photoionization

The molecular size dependence of primary fragmentation is studied for a series of β -naphthyl esters having alkyl chain lengths from C2 to C18. The esters are vaporized at a known ftemperature and ionized by coherent vacuum ultraviolet radiation at 10.5 eV. The photoionization wavelength is energetic enough to cause both metastable and nonmetastable primary fragmentation to m / z 144, but not energetic enough to cause secondary fragmentation to m / z 115 or 116. Under these conditions, the ratio of the nonmetastable-to-metastable daughter ion current, D/m _D ^′ is expected to give a rough indication of the average parent ion dissociation rate. The D / m _D ratio decreases with increasing molecular size, but not as quickly as expected by simple RRK theory. This behavior along with temperature dependence studies suggests that the internal energy required for dissociation is provided in substantial part by both the initial thermal internal energy and the energy imparted by the photoionization step. The role of thermal energy in the dissociation of large ions is discussed.     

Deuterium isotope effects on hydrophobic interactions: the importance of dispersion interactions in the hydrophobic phase

Hydrogen/deuterium isotope effects on hydrophobic binding were examined by means of reversed-phase chromatographic separation of protiated and deuterated isotopologue pairs for a set of 10 nonpolar and low-polarity compounds with 10 stationary phases having alkyl and aryl groups bonded to the silica surface. It was found that protiated compounds bind to nonpolar moieties attached to silica more strongly than deuterated ones, demonstrating that the CH/CD bonds of the solutes are weakened or have less restricted motions when bound in the stationary phase compared with the aqueous solvent (mobile phase). The interactions responsible for binding have been further characterized by studies of the effects of changes in mobile phase composition, temperature dependence of binding, and QSRR (quantitative structure-chromatographic retention relationship) analysis, demonstrating the importance of enthalpic effects in binding and differentiation between the isotopologues. To explain our results showing the active role of the hydrophobic (stationary) phase we propose a plausible model that includes specific contributions from aromatic edge-to-face attractive interactions and attractive interactions of aliphatic groups with the pi clouds of aromatic groups present as the solute or in the stationary phase.     

Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme

In this paper, we present a nanoscale study of the supramolecular structure of the dehydrogenate polymer (ZL-DHP) lignin model compound. The combination of near-field scanning optical microscopy (NSOM or SNOM) and atomic force microscopy (AFM) has been utilized to explore physicochemical properties of the lignin model compound on a scale ranging from individual macromolecules to globular supramolecular assemblies. By utilizing NSOM in transmission mode, the optical inhomogeneity in the lignin supramolecular structure has been observed for the first time. In particular, the transmission-mode NSOM images reveal a combination of hollow and layered supramolecular globular structure in the lignin model compound. Through the paired use of TappingMode and pulsed-mode AFM, we have also confirmed the existence of regions with different rheological properties on the single lignin model compound supramolecular assembly.