Pankaj Sehgal

α-Lactalbumin is unfolded by all classes of surfactants but by different mechanisms

We show that all four classes of surfactants (anionic, cationic, non-ionic, and zwitterionic) denature alpha-lactalbumin (alphaLA), making alphaLA an excellent model system to compare their denaturation mechanisms. This involves at least two steps in all surfactants but is more complex in charged surfactants due to their strong binding properties. At very low concentrations, charged surfactants bind specifically as monomers, but the first denaturation process only sets in when 4-10 surfactant molecules are bound to form clusters on the protein surface and is followed by a second loss of structure as 20-25 surfactant molecules are bound. Sub-micellar interactions can be modeled as simple independent binding at multiple sites which does not achieve saturation before micelle formation sets in. In contrast, no specific sub-micellar surfactant binding is detected by calorimetry in the presence of zwitterionic and non-ionic surfactants, and denaturation only occurs around the cmc. Unfolding rates are very rapid in charged surfactants and reach a similar plateau level around the cmc, indicating that monomers and micelles operate on a mutually exclusive basis. In contrast, unfolding occurs slowly in zwitterionic and non-ionic surfactants and the rate increases with the cmc, suggesting that monomers cooperate with micelles in denaturation.

Thermodynamics of unfolding of an integral membrane protein in mixed micelles

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl malto‐side facilitate reversible and quantitative unfolding and refolding. The 4‐transmembrane helix protein DsbB from the inner membrane of Escherichia coli unfolds in mixed micelles according to a three‐state mechanism involving an unfolding intermediate I. The temperature dependence of the kinetics of this reaction between 15° and 45°C supports that unfolding from I to the denatured state D is accompanied by a significant decrease in heat capacity. For water‐soluble proteins, the heat capacity increases upon unfolding, and this is generally interpreted as the increased binding of water to the protein as it unfolds, exposing more surface area. The decrease in DsbBs heat capacity upon unfolding is confirmed by independent thermal scans. The decrease in heat capacity is not an artifact of the use of mixed micelles, since the water soluble protein S6 shows conventional heat‐capacity changes in detergent. We speculate that it reflects the binding of SDS to parts of DsbB that are solvent‐exposed in the native DM‐bound state. This implies that the periplasmic loops of DsbB are relatively unstructured. This anomalous thermodynamic behavior has not been observed for β‐barrel membrane proteins, probably because they do not bind SDS so extensively. Thus the thermodynamic behavior of membrane proteins appears to be intimately connected to their detergent‐binding properties.

Interaction and Stability of Mixed Micelle and Monolayer of Nonionic and Cationic Surfactant Mixtures

Interaction and stability of binary mixtures of cationic surfactants hexadecyltrimethylammonium bromide (HTAB) or hexadecylpyridinium bromide (HPyBr) with nonionic surfactant decanoyl-N-methyl-glucamide (Mega-10) have been studied at different mole fraction of cationic surfactants by using interfacial tension measurements and fluorescence probe techniques. From interfacial tension measurements, the critical micellar concentration and various interfacial thermodynamic parameters have been evaluated. The experimental cmcs were analyzed with the pseudophase separation model, the regular solution theory, and the Maedas approach. These approaches allowed us to determine the interaction parameter and composition in the mixed state. By using the static quenching method, the mean micellar aggregation numbers of pure and mixed micelles of HTAB + Mega-10 were obtained. It has been observed that the aggregation number of mixed micelles deviates negatively from the ideal behavior. The micropolarity of the micelle was monitored with pyrene fluorescence intensity ratio and found to be increase with the increase of ionic content. The polarization of fluorescence probe Rhodamine B was monitored at different mole fraction of cationic surfactants.

pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus

Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4-6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately -12 to approximately -5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

Mixed Monolayer and Micelle Formation of Cationic and Zwitterionic Surfactant of Identical Hydrocarbon Tail in an Aqueous Medium: Interfacial Tension, Fluorescence Probe, Dynamic Light Scattering, and Viscosity Studies

We have studied the properties of the mixed monolayers and the mixed micelles formed from the mixture of the cationic surfactant dodecyltrimethylammonium bromide (DTAB) and the zwitterionic surfactant dimethyldodecylammoniopropanesulfonate (DPS), that have the identical hydrocarbon tail but different polar head groups, by measuring interfacial tension, fluorescence, dynamic light scattering (DLS), and viscosity. From the results of interfacial tension measurements, the various interfacial and bulk parameters such as the maximum surface excess, the surface pressure at the critical micellar concentration, and the standard free energies of interfacial adsorption and of micellization have been evaluated. The deviation from the ideal of the mixed micelles has been discussed on the basis of Clint theory. The interaction parameters in the mixed micelles and in the mixed monolayers, and also the compositions of the mixed micelles and the mixed monolayers were obtained with the help of regular solution theory. From a static fluorescence quenching method, the mean micellar aggregation number of pure and mixed micelles was obtained. Microviscosity in the pure and mixed micelles was monitored by fluorescence polarization measurements using Rhodamine B (RB) as a fluorescence probe. The hydrodynamic radii of the micelles were obtained in the mixtures at different mole fractions of surfactant from DLS measurements. The change of viscosity with α of DTAB revealed a minimum, and it was shifted to a higher αvalue in the mixture of high concentration of surfactant. This minima suggested a structural change in the mixed micelles.

Interactions of γ-Cyclodextrin with the Mixed Micelles of Anionic Surfactants and Their Inclusion Complexes Formation

Interactions of γ-cyclodextrin (γ-CD) with the single and mixed micelles of sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) have been studied at different concentrations of γ-CD by using conductivity measurements. From conductivity data, the pure and mixed critical micellar concentration (cmc), the equivalent ionic conductivities of the monomeric species (Λ m), the associated species (Λ assc) and the micelle (Λ mic), the degree of counterion dissociation (χ) in the presence of γ-CD have been evaluated from the slope of the conductivity versus concentration plots for the pure and binary mixture of surfactants. From the dependence of cmc of the surfactantson γ-CD concentration, we have deduced the association constant (K) of surfactant-γ-CD inclusion complexes assuming 2:1 stoichiometry. Theories of Clint, regular solution, and Motomuras have been used for the evaluation of ideality or nonideality of the mixed system. Mixed micelles were found to be rich in SDS content in the presence and the absence of γ-CD. The cmc values have been used to evaluate the transfer of standard free energy of micelles (ΔG0 M,tr) from the aqueous medium to additive medium.

Influence of β‐Cyclodextrin on the Mixed Micellization Process of Sodium Dodecyl Sulfate and Sodium Lauroyl Sarcosine and Formation of Inclusion Complexes

In this work, we have studied the influence of different concentrations of β‐Cyclodextrin (β‐CD) on the mixed micellization of anionic surfactants sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) at different SDS mole fractions (αSDS). From conductivity data, the critical micellar concentration (cmc), the equivalent ionic conductivities of the monomeric species (Λm), the associated species (Λassc) and the micelle (Λmic), the degree of counterion dissociation (α) in the presence of β‐CD were evaluated from the slope of the conductivity versus concentration plots for the pure and binary mixtures. The apparent cmc of the surfactants vary linearly with the β‐CD concentrations. From the dependence of cmc of the surfactants on β‐CD concentration, we have deduced the association constant (K) of surfactant‐β‐CD inclusion complexes assuming 1∶1 stoichiometry. Theories of Clint, Regular solution, and Motomuras have been used for the evaluation of ideality or nonideality of the mixed system. Mixed micelles were found to be rich in SDS content at the cmc in the presence and the absence of β‐CD. The cmc values have been used to evaluate the transfer of standard free energy of micelles (ΔG0 M,tr) from the aqueous medium to additive medium.

Interactions between the cationic surfactants bearing different polar head groups : Interfacial, conductivity, NMR, and fluorescence studies

Interfacial tension (γ), conductivity (κ), nuclear magnetic resonance (NMR), and fluorescence measurements have been carried out to study the mixed interfacial and micellar behavior of cationic surfactants cetyltributylphosphonium bromide (CTBB) and the cetyltrimethylammonium bromide (CTAB). From the γ versus log C s plots, the values of critical micellar concentration (cmc) and various interfacial parameters were computed. From κ measurements, the equivalent conductivities of the monomers (Λ mon), the micelles (Λ mic) states and the degree of counterion dissociation (δ) have been evaluated. The cmc values have been analyzed in the context of the pseudophase separation model and regular solution theory. The interaction parameters, βm and βσ, in the mixed micelle as well as in the mixed monolayer, respectively, also have been computed. The self‐diffusion coefficients for the micelles have been evaluated by using NMR spectroscopy. From the fluorescence quenching method, the mean micellar aggregation number (N agg) of the pure and mixed micelles has been obtained from the slope of the ratio of fluorescence intensities in the absence and in the presence of quencher (ln (I 1,0/I 1) versus [Q] plots. It was found that the incorporation of CTBB into the mixed micelle decreases the N agg. The microviscosity of the fluorescence probe Rhodamine (RB) was monitored by using fluorescence polarization measurements. The values of fluorescence anisotropies (r) indicate that the penetration of CTBB monomer into CTAB micelles produced less rigid mixed micelles.

Aggregated assemblies of sodium dodecyl sulfate/dimethyldodecyl ammoniopropane sulfonate and phospholipids at the interface and in the bulk.

Interactions between the binary combinations of sodium dodecyl sulfate (SDS) or dimethyldodecylammoniopropane sulfonate (DPS) with L-alpha-phosphatidylcholine (PC), 1,2-didecanoyl-sn-glycero-3-phosphacholine (DPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphacholine (PPC) at the air/water interface and in aqueous bulk were evaluated with the help of interfacial tension (gamma) and pyrene fluorescence (I(1)/I(3)) measurements by studying the aggregation processes of SDS and DPS in pure water and in the presence of 7-36 microM of each lipid. The gamma measurements suggested that the interface was mainly occupied by the surfactant monomers especially in the presence of PC and PPC, and the surfactant-PC or surfactant-PPC aggregates were mainly available in the bulk with the least surface activity. Significant surface activity was observed in the case of a surfactant-DPC complex. The fluorescence measurements showed clear onset, C(1), and completion, C(2), of a vesicles solubilization process upon incorporation of surfactant monomers into the vesicles in the presence of DPC and PPC whereas this process was not visible in the presence of PC. A comparative study of all the three lipids indicated that both PC and PPC were mainly available in the aggregated form in the bulk due to their higher hydrophobicities and, hence, were the least surface active. On the other hand, DPC with relatively lower hydrophobicity showed considerable surface activity even in the monomeric form. Among both surfactants, DPS showed stronger interactions with DPC and PPC in comparison to SDS due to its zwitterionic nature, which could easily accommodate itself into the lipid-aggregated assemblies with similar headgroup natures, and helped in reducing the interhead-group repulsions.

Erratum to “Aggregated Assemblies of Sodium Dodecyl Sulfate/Dimethyldodecyl Ammoniopropane Sulfonate and Phospholipids at the Interface and in the Bulk”: [J. Colloid Interface Sci. 252 (2002) 195–201]

Abstract Interactions between the binary combinations of sodium dodecyl sulfate (SDS) or dimethyldodecylammoniopropane sulfonate (DPS) with L -α-phosphatidylcholine (PC), 1,2-didecanoyl- sn -glycero-3-phosphacholine (DPC), and 1,2-dipalmitoyl- sn -glycero-3-phosphacholine (PPC) at the air/water interface and in aqueous bulk were evaluated with the help of interfacial tension (γ) and pyrene fluorescence ( I 1 / I 3 ) measurements by studying the aggregation processes of SDS and DPS in pure water and in the presence of 7–36 μM of each lipid. The γ measurements suggested that the interface was mainly occupied by the surfactant monomers especially in the presence of PC and PPC, and the surfactant–PC or surfactant–PPC aggregates were mainly available in the bulk with the least surface activity. Significant surface activity was observed in the case of a surfactant–DPC complex. The fluorescence measurements showed clear onset, C 1 , and completion, C 2 , of a vesicles solubilization process upon incorporation of surfactant monomers into the vesicles in the presence of DPC and PPC whereas this process was not visible in the presence of PC. A comparative study of all the three lipids indicated that both PC and PPC were mainly available in the aggregated form in the bulk due to their higher hydrophobicities and, hence, were the least surface active. On the other hand, DPC with relatively lower hydrophobicity showed considerable surface activity even in the monomeric form. Among both surfactants, DPS showed stronger interactions with DPC and PPC in comparison to SDS due to its zwitterionic nature, which could easily accommodate itself into the lipid-aggregated assemblies with similar headgroup natures, and helped in reducing the interheadgroup repulsions.