Effect of salt concentration and pH on the hydrophobicity parameters of surfactants studied by TLC and spectral mapping technique

The lipophilicity and specific hydrophobic area of 56 surfactants having different hydrophobic moiety and different length of ethylene oxide chain were determined by reversed-phase thin-layer chromatography and the strength and selectivity of the effect of sodium chloride and pH on the hydrophobicity parameters was elucidated using spectral mapping technique followed by two-dimensional nonlinear mapping. In each instance significant linear correlations were found between the lipophilicity and specific hydrophobic surface area of surfactants suggesting that from a chromatographic point of view they behave as a homologous series of solutes. It was established that the strength of the effect of both salt concentration and pH is relatively low and the selectivity of their influence on the hydrophobicity parameters is markedly different.     

Influence of salt and pH on the hydrophobicity parameters of antisense nucleosides studied by reversed-phase thin-layer chromatography and multivariate mathematical-statistical methods.

The lipophilicity and specific hydrophobic surface area of 12 8-substituted 2'-deoxyadenosine and 17 5-substituted-2'-deoxyuridine derivatives were determined by reversed-phase thin-layer chromatography in ion-free eluents and in eluents containing sodium chloride, sodium acetate and acetic acid. The strength and selectivity of the effect of eluent additives were separated by use of spectral mapping technique followed by two-dimensional nonlinear mapping. The relationship between the structural characteristics and hydrophobicity parameters was elucidated by stepwise regression analysis. Eluent additives exert a considerable influence on both hydrophobicity parameters. The effect of sodium chloride and acetic acid was higher than that of sodium acetate. The strength and selectivity of the sensitivity of nucleosides towards eluent additives significantly depended on the character of the ring structure and on the length of the apolar alkyl chain. The influence of the degree of unsaturation and the branching of the alkyl substituent was negligible.     

Relationship between the physicochemical parameters and biological activity of sulfosuccinic acid ester surfactants

The effect of 11 sulfosuccinic acid ester surfactants on the abiotrophic developmental forms of the asexual spores of Plasmopara halstedii was determined. The strength and the selectivity of the effect was separated by the spectral mapping technique and the relationship between the biological activities and physico-chemical parameters of anionic surfactants was elucidated by stepwise regression analysis. It was established that the plasmalemma of cell wall less zoospores showed the highest and the resting zoosporangia the lowest average sensitivity toward the surfactants. The biological efficacy of surfactants was highly different the di-n-octyl ester being the most effective. Calculations proved that both the strength and selectivity of the biological activity mainly depends on the lipophilicity of the molecule and, to a lesser extent, on the electronic parameters.     

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.     

Structural analysis of lipid complexes of GM2-activator protein

The GM2-activator protein (GM2-AP) is a small lysosomal lipid transfer protein essential for the hydrolytic conversion of ganglioside GM2 to GM3 by beta-hexosaminidase A. The crystal structure of human apo-GM2-AP is known to consist of a novel beta-cup fold with a spacious hydrophobic interior. Here, we present two new structures of GM2-AP with bound lipids, showing two different lipid-binding modes within the apolar pocket. The 1.9A structure with GM2 bound shows the position of the ceramide tail and significant conformational differences among the three molecular copies in the asymmetric unit. The tetrasaccharide head group is not visible and is presumed to be disordered. However, its general position could be established through modeling. The structure of a low-pH crystal, determined at 2.5A resolution, has a significantly enlarged hydrophobic channel that merges with the apolar pocket. Electron density inside the pocket and channel suggests the presence of a trapped phospholipid molecule. Structure alignments among the four crystallographically unique monomers provide information on the potential role for lipid binding of flexible chain segments at the rim of the cavity opening. Two discrete orientations of the S130-T133 loop define an open and a closed configuration of the hydrophobic channel that merges with the apolar pocket. We propose: (i) that the low-pH structure represents an active membrane-binding conformation; (ii) that the mobile S130-T133 loop serves as a gate for passage of ligand into the apolar pocket; and (iii) that this loop and the adjacent apolar V59-W63 loop form a surface patch with two exposed tryptophan residues that could interface with lipid bilayers.     

Peroxyoxalate chemiluminescence enhanced by oligophenylenevinylene fluorophores in the presence of various surfactants

The effect of several surfactants on peroxyoxalate chemiluminescence (PO‐CL) using oligophenylenevinylene fluorophores was investigated. Among several oligophenylenevinylenes consisting of stilbene units, linearly conjugated ones, such as distyrylbenzene and distyrylstilbene, effectively enhanced PO‐CL efficiency. Various effects of anionic, cationic, amphoteric and non‐ionic surfactants on the CL efficiency of PO‐CL were determined using three oxalates and the distyrylbenzene fluorophore. Anionic and non‐ionic surfactants effectively enhanced CL efficiency, in contrast to the negative effect of cationic and amphoteric surfactants. Non‐ionic surfactants were also effective in CL reactions of oxalates bearing dodecyl ester groups by the hydrophobic interaction between their alkyl chains. Considering these results, the surfactants not only increase the concentrations of water‐insoluble interacting species in the hydrophobic micelle cores, but also control rapid degradation of the oxalates by alkaline hydrolysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrophobic interaction of human, mouse, and rabbit interferons with immobilized hydrocarbons.

Interferons of human, mouse, and rabbit origin bind to straight chain hydrocarbons immobilized on agarose. The hydrophobic nature of binding is established by the following observations: (a) a positive correlation between the length of hydrocarbon ligand and the strength of interaction; (b) a stronger interaction with hydrocarbon ligands terminated with apolar rather than polar head groups; (c) a lack of dependence of binding on ionic strength and pH of the solvent; (d) a reversal of binding by ethylene glycol, a hydrophobic solute; (e) an increasing eluting efficacy of tetraalkylammonium ions with the length of their alkyl substituents. The hydrophobic interactions of human interferon underlie the efficiency of two-step chromatographic procedures. For example, human embryo kidney interferon can be purified about 3,600-fold by sequential chromatography on (a) concanavalin A-agarose, (b) octyl-agarose. Another two-step procedure: (a) concanavalin A-agarose, (b) L-tryptophan-agarose, gives about 10,000-fold purification. The overall recovery of interferon in both cases in close to 90%.     

Adsorption properties of polar/apolar inducers at a charged interface and their relevance to leukemia cell differentiation.

The interfacial adsorption properties of polar/apolar inducers of cell differentiation (PAIs) were studied on a mercury electrode. This study, on a clean and reproducible charged surface, unraveled the purely physical interactions among these compounds and the surface, apart from the complexity of the biological membrane. The interfacial behavior of two classical inducers, hexamethylenebisacetamide (HMBA) and dimethylsulfoxide, was compared with that of a typical apolar aliphatic compound, 1-octanol, that has a similar hydrophobic moiety as HMBA but a much smaller dipolar moment. Both HMBA and Octanol adsorb flat in contact with the surface because of hydrophobic forces, with a very similar free energy of adsorption. However, the ratio of polar to apolar moieties in PAIs turned out to be crucial to drive the adsorption maximum toward physiological values of surface charge density, where octanol is desorbed. The electrostatic effects in the interfacial region reflected the adsorption properties: the changes in the potential drop across the interfacial region as a function of the surface charge density, in the physiological range, were opposite in PAIs as compared with apolar aliphatic compounds, as exemplified by octanol. This peculiar electrostatic effect of PAIs has far-reaching relevance for the design of inducers with an adequate therapeutic index to be used in clinical trials.     

The internal signal sequence of Escherichia coli leader peptidase is necessary, but not sufficient, for its rapid membrane assembly

Leader peptidase of Escherichia coli, a protein of 323 residues, has three hydrophobic domains. The first, residues 1-22, is the most apolar and is followed by a polar region (23-61) which faces the cytoplasm. The second hydrophobic domain (residues 62-76) spans the membrane. The third hydrophobic domain, which has a minimal apolar character, and the polar, carboxyl-terminal two-thirds of the protein are exposed to the periplasm. Deletion of either the amino terminus (residues 4-50) or the third hydrophobic region (residues 83-98) has almost no effect on the rate of leader peptidase membrane assembly, while the second hydrophobic domain is essential for insertion (Dalbey, R., and Wickner, W. (1987) Science 235, 783-787). To further define the roles of these domains, we have replaced the normal, cleaved leader sequence of pro-OmpA and M13 procoat with regions containing either the first or second apolar domain of leader peptidase. The second apolar domain supports the translocation of OmpA or coat protein across the plasma membrane, establishing its identity as an internal, uncleaved signal sequence. In addition to this sequence, we now find that leader peptidase needs either the amino-terminal domain or the third hydrophobic domain to permit its rapid membrane assembly. These results show that, although a signal sequence is necessary for rapid membrane assembly of leader peptidase, it is not sufficient.     

The use of a PMR probe for studying the hydrophobic properties of micrococcus lysodeikticus membranes and membrane fractions

The hydrophobic character of the trimethyl group of sodium 2,2-dimethyl-2-silapentane-5-sulfonate, makes it an effective PMR probe for apolar sites on proteins and membranes. By comparing the spin-spin relaxation rates of the free and bound probe the extent and strength of the interaction can be qualitatively compared for bovine serum albumin, membranes from Micrococcus lysodeikticus and for different fractions isolated from this membrane. It is concluded that this membrane has hydrophobic sites on or near its surface and that the number of such sites is sensitive to the ionic composition and to the pH of its aqueous environment. Removal of lipid from the membrane greatly increased the binding of the probe, while vesicular preparations of the lipid fraction itself gave no evidence of an interaction with the probe. The results are discussed in terms of protein-lipid-water interactions.     

Hydrophobic patches on the surfaces of protein structures

A survey of hydrophobic patches on the surface of 112 soluble, monomeric proteins is presented. The largest patch on each individual protein averages around 400 Ų but can range from 200 to 1,200 Ų. These areas are not correlated to the sizes of the proteins and only weakly to their apolar surface fraction. Ala, Lys, and Pro have dominating contributions to the apolar surface for smaller patches, while those of the hydrophobic amino acids become more important as the patch size Increases. The hydrophilic amino acids expose an approximately constant fraction of their apolar area independent of patch size; the hydrophobic residue types reach similar exposure only in the larger patches. Though the mobility of residues on the surface is generally higher, it decreases for hydrophilic residues with Increasing patch size. Several characteristics of hydrophobic patches catalogued here should prove useful in the design and engineering of proteins. © 1996 Wiley-Liss, Inc.     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

Multiple Determinants Direct the Orientation of Signal–Anchor Proteins: The Topogenic Role of the Hydrophobic Signal Domain

The orientation of signal–anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH₂-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7–25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH₂ terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH₂-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.     

Sequence-specific 1H NMR assignments and secondary structure of the streptococcal protein G B2-domain.

Two-dimensional NMR spectroscopy has been used to obtain sequence-specific 1H NMR assignments for the IgG-binding B2-domain of streptococcal protein G. Secondary structure elements were identified from analysis of characteristic backbone-backbone NOE patterns and amide proton exchange data. The B2-domain contains a four-stranded beta-sheet region in which the two inner strands form a parallel beta-sheet with each other and antiparallel beta-sheets with the outer strands. The outer strands are connected via a 16-residue alpha-helix and short loops on both ends of the helix. The alpha-helix and beta-sheet structures contain well-defined polar and apolar sides, and numerous long-range NOEs from the apolar helix to apolar sheet regions were used to derive a model for the global fold of the B2-domain. While the overall fold is similar to that obtained for B1-type domains, differences in amide proton exchange rates and hydrophobic packing are observed.     

Hydrogen evolution from dithionite and H2 photoproduction by hydrogenase incorporated into various hydrophobic matrices

The effects of surfactants, lipids and amphiphilic viologen mediators on H2 production from dithionite as well as on Ru(bpy) sensitized H2 photoproduction by hydrogenase from Thiocapsa roseopersicina was studied. Three systems which differed as to the nature of the hydrophobic matrix around the hydrogenase were tested. An enhanced hydrogenase activity was observed in the presence of surfactants, in the 1-6 mM concentration range. Hydrogenase showed a selectivity for the amphiphilic viologens, 2C7-diCl was the most efficient electron mediator in both reactions. H2 photoproduction seemed not to be feasible in the detergent-hydrogenase system because of intensive foaming. Hydrogenase incorporated into liposomes catalyzed H2 photoevolution efficiently but the rate was decreasing in time, though reversibly. Using intact bacterial cells instead of purified hydrogenase yielded stable H2 photoevolution for at least 12 hours. This system offers several advantages for potential practical applications.     

Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Problems inherent in the isolation of thiols from natural sources, such as oxidation, undesirable addition reactions, and low concentration of thiol species in cell-free extracts, can be circumvented by reversible derivatization to a less labile form which can be concentrated selectively. These objectives are realized by converting thiols to heterodisulfides in which the thiol partner is an apolar thiol with strong affinity for hydrophobic stationary phases. When reacted with 2-S-(2(')-thiopyridyl)-6-hydroxynaphthyldisulfide at pH<5, where most thiol species are relatively stable to atmospheric oxidation, mixed disulfides with 2-mercapto-6-hydroxynaphthalene as the apolar partner are obtained in good yield and can be concentrated onto a hydrophobic stationary phase. Such heterodisulfides exhibit excellent chromatographic properties when separated on reversed-phase media and the derivatization reaction can, therefore, be conveniently monitored. Following their isolation as the heterodisulfides the thiol species of interest are recovered by reduction and facile separation from the apolar 2-mercapto-6-hydroxynaphthalene partner.     

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.     

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.     

Surface hydrophobic groups, stability, and flip-flopping in lattice proteins

Two-dimensional lattice protein models were studied in two approximations of the conformational equilibrium to elucidate the role of surface hydrophobic groups in their stabilities. We demonstrate that stability of any compactly folded sequence is determined by its ability to "flip-flop" (refold) into alternative compact structures. The degree of stability required for folded sequences determines the average numbers of surface hydrophobic groups in stable lattice structures which are in good agreement with ratios of core to surface hydrophobic groups in real proteins. However, the average destabilization of the native structure per surface hydrophobic group is small (0-0.25 kcal/mol), often disagrees with the free energies derived from the ratios of core to surface hydrophobic groups in the same structures, and has a combinatorial entropic nature independent of the strength of structure stabilizing interactions. This suggests that the free energies derived from the core to surface ratios of hydrophobic groups in real proteins have little to do with folding thermodynamics. On average, sequences with highly stable native structures are the least hydrophobic. The results suggest that in designing novel stable proteins hydrophobic groups on the surface should be avoided to reduce the possibility of flip-flopping. The average stability of highly designable structures is never higher than that of some low designability structures, contrary to the accepted view. In the equilibrium approximation with alternative compact and partially unfolded structures, the requirement of high stability selects a unique 5 x 5 structure formed by only a few sequences, suggesting much stronger sequence selectivity than commonly thought.     

Derivatives of glutamic acid as new surfactants>

Summary. Starting from glutamic acid, different types of surfactants have been synthesised by using original trimodular strategies. Monosubstituted zwitterionic amides of glutamic acid obtained with excellent yields show good surface activity. The grafting of a second hydrophobic side-chain leads to bicatenar cationic surfactants or to disubstituted nonionic cyclic compounds. In order to reduce the hydrophobic character of the bicatenar surfactants, a second synthetic method has been developed, allowing the introduction of a polar sugar group into these molecules. The surfactant properties of several of the products have been determined by physico-chemical methods such as surface tension measurements and compression isotherm studies by means of a Langmuir balance.