Anionic branched surfactants as alternative denaturing agents for protein separations.

Denaturation of a group of model proteins of diverse size and composition with three branched alkyl surfactants-sodium 2-ethylhexyl sulfate (2-EHS), sodium 3,7-dimethyloctyl sulfate (3,7-DMOS), and sodium 2-butyloctyl sulfate (2-BOS)-has been investigated using circular dichroism (CD), small-angle X-ray scattering, and polyacrylamide gel electrophoresis (PAGE). Circular dichroism reveals that 2-BOS disrupts to a higher extent the secondary structure for most of the proteins. Also, it is found that upon adsorption the shape of the protein-surfactant complexes varies from "pearl necklace" to ellipsoidal depending on the surfactant that is used. Macroscopic separations also reveal that branching sodium alkyl sulfates with n-butyl (2-BOS) and n-methyl (3,7-DMOS) groups significantly affects their performance in PAGE. 3,7-DMOS and 2-BOS result in anomalous migrations that deviate from the expected electrophoretic mobility. A combined interpretation of spectroscopy, scattering, and polyacrylamide gel electrophoresis suggests that 2-BOS promotes stronger modification of proteins during denaturation. The findings in this work aim to improve protein electrophoretic separations and the design of novel surfactants.

Flow-induced structured phase in nonionic micellar solutions.

In this work, we consider the flow of a nonionic micellar solution (precursor) through an array of microposts, with focus on its microstructural and rheological evolution. The precursor contains polyoxyethylene(20) sorbitan monooleate (Tween-80) and cosurfactant monolaurin (ML). An irreversible flow-induced structured phase (NI-FISP) emerges after the nonionic precursor flows through the hexagonal micropost arrays, when subjected to strain rates ~10(4) s(-1) and strain ~10(3). NI-FISP consists of close-looped micellar bundles and multiconnected micellar networks as evidenced by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). We also conduct small-angle neutron scattering (SANS) measurements in both precursor and NI-FISP to illustrate the structural transition. We propose a potential mechanism for the NI-FISP formation that relies on the micropost arrays and the flow kinematics in the microdevice to induce entropic fluctuations in the micellar solution. Finally, we show that the rheological variation from a viscous precursor solution to a viscoelastic micellar structured phase is associated with the structural evolution from the precursor to NI-FISP.

Worming their way into shape: toroidal formations in micellar solutions.

We report the formation of nanostructured toroidal micellar bundles (nTMB) from a semidilute wormlike micellar solution, evidenced by both cryogenic-electron microscopy and transmission electron microscopy images. Our strategy for creating nTMB involves a two-step protocol consisting of a simple prestraining process followed by flow through a microfluidic device containing an array of microposts, producing strain rates in the wormlike micelles on the order of 10(5) s(-1). In combination with microfluidic confinement, these unusually large strain rates allow for the formation of stable nTMB. Electron microscopy images reveal a variety of nTMB morphologies and provide the size distribution of the nTMB. Small-angle neutron scattering indicates the underlying microstructural transition from wormlike micelles to nTMB. We also show that other flow-induced approaches such as sonication can induce and control the emergence of onion-like and nTMB structures, which may provide a useful tool for nanotemplating.

Structural analysis of protein denaturation with alkyl perfluorinated sulfonates.

The denaturation of three model proteins, bovine serum albumin (BSA), ovalbumin, and lysozyme, with fully fluorinated potassium sulfonate surfactants was studied using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), polyacrylamide gel electrophoresis (PAGE), and circular dichroism (CD). Scattering analysis revealed that protein-surfactant complexes of fluorinated molecules could also organize into pearl-necklace structures. In contrast, fluorinated surfactants with just four carbons in the tail (PFC(4)S) promoted the formation of much larger aggregates. The changes in secondary structure of the protein-surfactant complexes were also analyzed by CD to determine the relative content of α-helix and ß-sheet motifs. The CD spectra suggest that the α-helix content steadily decrease when proteins were denatured with surfactants of smaller tail lengths. Potassium perfluoro-octanesulfonate (PFC(8)S) was able to form stable complexes with all three of the model proteins, but lysozyme was unstable when denatured in the presence of shorter fluorinated surfactants. Finally, PFC(8)S was also found to be more effective in PAGE separations than a similarly sized hydrogenated surfactant, sodium octyl sulfate (SC(8)S), but fluorinated surfactants with only six (PFC(6)S) or four (PFC(4)S) carbons in the tail length showed much weaker performance in proteomic separations.

Numerical validation of IFT in the analysis of protein-surfactant complexes with SAXS and SANS.

The use of the indirect Fourier transform methods for evaluating structural parameters directly in real space with small-angle scattering measurements is validated for the analysis of protein-surfactant complexes. An efficient Monte Carlo approach rapidly generates in silico structures based on a realistic pearl-necklace model for denatured proteins decorated with surfactant micelles. Corresponding scattering profiles are calculated and averaged over a large number of possible configurations for each structure. IFT algorithms are then used to calculate the corresponding pair-distance distribution function, and structural information is extracted directly without model fitting. The extracted parameters are compared and correlated with the known structure of the simulated complexes to assess the quality of the information that can be reliably obtained from these systems. The average extension, nearest-neighbor micelle distance, and average number of associated micelles are all accurately extracted through IFT calculations. Improved and simple approaches to reliably extract the average extension of the complex and the total number of associated micelles are presented.

Competitive adsorption of thiolated poly(ethylene glycol) and alkane-thiols on gold nanoparticles and its effect on cluster formation.

The surface concentration and conformation of thiol-terminated poly(ethylene glycol) (PEG) on gold nanoparticles are studied before and after coadsorption of alkane-thiols. Thermogravimetric analysis (TGA) indicates alkane-thiol ligands will competitively adsorb on gold surfaces of nanoparticles and that the extent of PEG-thiol replacement depends on the specific length of the alkane-thiol molecule. The conformation of the polymer is also affected by the length and packing density of the alkane-thiol. Dynamic light scattering (DLS) shows that the hydrodynamic size of coated particles has an intermediate maximum for the adsorption of octane-thiol, which also forms the most densely packed alkane-thiol monolayers. These two factors greatly impact the formation of clusters by nanoparticle surfactants. Small angle X-ray scattering (SAXS) shows that the largest clusters are formed when particles have a low PEG-thiol surface concentration and an extended PEG conformation.

Pickering emulsions stabilized by nanoparticle surfactants.

Amphiphilic gold nanoparticles are demonstrated to effectively stabilize emulsions of hexadecane in water. Nanoparticle surfactants are synthesized using a simple and scalable one-pot method that involves the sequential functionalization of particle surfaces with thiol-terminated polyethylene glycol (PEG) chains and short alkane-thiol molecules. The resulting nanoparticles are shown to be highly effective emulsifying agents due to their strong adsorption at oil-water and air-water interfaces. The original nonfunctionalized gold nanoparticles are unable to effectively stabilize oil-water emulsions due to their small size and low adsorption energy. Small-angle X-ray scattering and electron microscopy are used to demonstrate the formation of nanoparticle-stabilized colloidosomes that are stable against coalescence and show significant shifts in plasmon resonance enhancing the near-infrared optical absorption.

SANS and SAXS analysis of charged nanoparticle adsorption at oil-water interfaces.

A systematic study of the adsorption of charged nanoparticles at dispersed oil-in-water emulsion interfaces is presented. The interaction potentials for negatively charged hexadecane droplets with anionic polystyrene latex particles or cationic gold particles are calculated using DLVO theory. Calculations demonstrate that increased ionic strength decreases the decay length of the electrostatic repulsion leading to enhanced particle adsorption. For the case of anionic PS latex particles, the energy barrier for particle adsorption is also reduced when the surface charge is neutralized through changes in pH. Complementary small-angle scattering experiments show that the highest particle adsorption for PS latex occurs at moderate ionic strength and low pH. For cationic gold particles, simple DLVO calculations also explain scattering results showing that the highest particle adsorption occurs at neutral pH due to the electrostatic attraction between oppositely charged surfaces. This work demonstrates that surface charges of particles and oil droplets are critical parameters to consider when engineering particle-stabilized emulsions.

Fibrin clot structure and mechanics associated with specific oxidation of methionine residues in fibrinogen.

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, ß, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.

Aqueous dispersions of colloidal poly(3-hexylthiophene) gel particles with high internal porosity.

This work outlines the development of nano-porous, sub-micron poly(3-hexylthiophene) (P3HT) gel particles as solution-processable inks for applications in polymer solar cells. These dispersions are produced by emulsifying bulk P3HT organogels into water containing surfactant. The optical characteristics and stability of the resulting gel particles are assessed and their structure characterized. The P3HT within the gel particles is shown to retain its crystallinity with no evidence of doping. The gel particles are shown to be stable against aggregation due to the presence of surfactant at the oil/water interface. The fracture of the gel network during emulsification produces a bimodal distribution of particles that increase in size with increasing P3HT concentration in the 'parent' organogel. Small Angle Neutron Scattering measurements show that the particles maintain the structure of the bulk gels with high specific surface area. Spray-coating the gel particle dispersions produces uniform thin-films, which have been used to fabricate polymer/fullerene solar cells with a fully spray-coated active layer.

The conformation of the poly(ethylene glycol) chain in mono-PEGylated lysozyme and mono-PEGylated human growth hormone.

Covalent conjugation of poly(ethylene glycol) or "PEGylation" has proven an effective strategy to improve pharmaceutical protein efficacy by hindering recognition by proteases, inhibitors, and antibodies and by retarding renal clearance. Because it determines the strength and range of intermolecular steric forces and the hydrodynamic properties of the conjugates, the configuration of protein-conjugated PEG chains is the key factor determining how PEGylation alters protein in vivo circulation time. Mono-PEGylated proteins are typically described as having a protective PEG shroud wrapped around the protein, but recent dynamic light scattering studies suggested that conjugates adopt a dumbbell configuration, with a relatively unperturbed PEG random coil adjacent to the globular protein. We used small-angle neutron scattering (SANS) to distinguish between the dumbbell model and the shroud model for chicken-egg lysozyme and human growth hormone covalently conjugated to a single 20 kDa PEG chain. The SANS contrast variation technique was used to isolate the PEG portion of the conjugate. Scattering intensity profiles were well described by the dumbbell model and inconsistent with the shroud model.

Structural analysis of protein complexes with sodium alkyl sulfates by small-angle scattering and polyacrylamide gel electrophoresis.

Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations.

Small angle scattering model for Pickering emulsions and raspberry particles.

Pickering emulsions, raspberry particles and other colloidal particle complexes are often characterized using small angle scattering techniques. The present work derives an analytical scattering model that accounts for the self-correlation of a spherical core and surface adsorbed particles as well as the particle-particle and core-particle correlation terms characteristic of Pickering emulsions and raspberry particles. It is shown that contrast matching of the scattering length density is not essential to obtain meaningful information as long as the scattering contrasts of all phases are precisely known. The derived equations are useful for analyzing data and planning experiments for Small Angle Neutron Scattering (SANS) and Small Angle X-ray Scattering (SAXS) involving these colloidal systems.

Neutron-scattering probe of complexes of sodium dodecyl sulfate and serum albumin during polyacrylamide gel electrophoresis.

Small-angle neutron scattering (SANS) is used to probe the conformation of SDS-BSA protein surfactant complexes during electrophoresis in cross-linked polyacrylamide gels. Contrast variation permits independent probing of the structure of protein-surfactant complexes with negligible scattering contributions from the polyacrylamide matrix. The conformation of the protein complexes in the gel is found to be independent of the electric fields that are applied in this work (10 V/cm). Furthermore, there are no signs of large-scale macromolecular orientation (anisotropy) in the scattering patterns. However, the scattering shows that there are significant interparticle correlations between the protein-surfactant complexes that are electrophoretically inserted into the gel. These interactions develop when the total concentration of protein in the gels reaches values that are larger than approximately 1 mg/mL. The correlations are due to molecular crowding in the small fraction of pores that are available for protein migration.