Alterations in the Detergent-Induced Membrane Permeability and Solubilization of Saturated Phosphatidylcholine/Cholesterol Liposomes: Effects of Poly(ethylene glycol)-Conjugated Lipid

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.     

LIPOSOME MICROENCAPSULATIONS WITHOUT USING ANY ORGANIC SOLVENT

Liposomes are microscopic vesicles composed of phospholipid bilayers surrounding discrete aqueous compartment. Eventhough phospholipid bilayers are formed spontaneously in water, their conventional preparation methods involve either organic solvents or detergent molecules. Organic solvents may create a serious safety problem during liposome manufacturing especially on a large scale and can cause denaturation of proteins and affect the membrane properties. Liposomes are very promising carriers for protein drugs which have aroused great interests in the field of pharmaceutics in these recent years.

The objective of this study was to develop a new dispersion process to prepare liposomes without the usage of organic solvents and detergents. The approach involved the usage of a microfludizer for the extrusion and homogenization of aqueous dispersion of phospholipids, and a modified evaporator in order to increase lipid thin film surface area. The properties of the liposomes prepared by the novel method were superior to the liposomes prepared by conventional methods.     

Process of destruction of large unilamellar vesicles by a nonionic detergent,octylglucoside, and size growth factor in vesicle formation from phospholipid–detergent mixed micelles

The process of vesicle solubilization and size growth by detergents, especially by octylglucoside, was examined in detail in order to elucidate the phenomena observed in the vesicle-to-micelle transition and to clarify the size-determining factor of vesicles prepared by removing detergent from phospholipid–detergent mixed micelles. In the vesicle solubilization process, when the detergent concentration in the vesicle membrane reached a critical value, the collapse of large unilamellar vesicles (LUV) into small unilamellar vesicles (SUV) was observed. This newly appeared SUV were named SUV*. The SUV* could be produced by adding an appropriate amount of detergent to the SUV prepared by an ultrasonication method so as to increase the concentration to a little over the critical value, such as, in the case of adding octylglucoside, a molar ratio of 1.0–1.1 to phospholipid in the membrane phase. The SUV* containing octylglucoside were fusible and grow time-dependently, but those containing sodium cholate were not fusible. On the basis of the SUV* data, the following problems were solved: the variety of the size of the vesicles prepared by detergent removal from mixed micelles composed of a phospholipid and different detergents, or by different removal methods; the complex appearance of turbidity or vesicle size observed in vesicle destruction and formation; the conflict between LUV and SUV in the partition behavior of detergent and the size change with addition of detergent.     

Colorimetric determination of anionic detergents

Many types of anionic detergents can be determined colorimetrically by the action of the detergent on a protein-bromcresol purple complex. The amount of free dye liberated from the complex is proportional to the amount of detergent added between certain limits.     

A more reliable detergent for constant load experiments on polyethylene

Testing polyethylene materials for stress cracking resistance at elevated temperatures in the presence of a detergent solution is relatively popular. The detergents used for these tests are—to our knowledge—only of the ethoxylated nonylphenol type (trade names: Igepal, Antharox, Arkopal). Extensive investigations of solutions of these nonionic detergents used at 80 °C have shown that they are very susceptible to oxidation. Oxidation has already started after about one day at 80 °C and leads to erroneous failure times of polyethylene test bars exposed to the detergent and to an unacceptably large scatter in the data.

An alternative anionic alkylbenzene sulphonate (ABS) detergent has been tested which, when dissolved in drinking water, produces reproducible failure times of welded polyethylene samples, if the age of the detergent solution does not exceed about 2000 h at 80 °C.     

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d -maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor, and its complex with G_s protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.     

Influence of detergent additives on mobility of native and subviral rhinovirus particles in capillary electrophoresis

The electrophoretic properties of two human rhinovirus (HRV) serotypes, HRV2 and HRV14, their subviral particles, and their capsid proteins were investigated by CE using borate buffer, pH 8.3, as BGE and three different detergents as additives. In addition, the influence of modification of the capsid with an amine reactive fluorescent dye, Cy3.5, on migration in the electric field was assessed. We found that the reproducibility of the electrophoretic results was decisively dependent on the presence of the detergents above their respective CMC. As compared to the strong ionic detergent SDS, the nonionic, mild detergent dodecylpoly(ethyleneglycol ether) (D-PEG) efficiently and reproducibly resolved both, native viruses as well as subviral particles. Most of the analytes behaved as expected except native HRV2; this serotype showed a dramatically higher anionic mobility in SDS than in D-PEG. Additionally, its mobility decreased when each positive charge contributed from a lysine at the capsid surface was substituted by four negative charges upon derivatization with Cy3.5. We discuss the possibility that this effect is caused by differences in number and in arrangement of exposed lysines in the two serotypes leading to differences in the amount of bound SDS micelles.     

Regulation of CD40 reconstitution into a liposome using different ratios of solubilized LDAO to lipids

The integral membrane protein CD40 was found on the surface of B lymphocytes that interact with CD40L on T cells during the immune response. The hydrophobic transmembrane domains of membrane proteins can be stabilized in detergent or in lipid bilayers such as liposomes. Membrane proteins can be incorporated into the liposome in a similar fashion to the way they are handled in vivo. In this study, a large amount of full-sequence CD40 was produced using a bacterial system that contained a Mistic construct. The CD40 was then reconstituted into liposomes by detergent-mediated reconstitution. All stages in the process of liposome disruption with various detergent ratios were easily observed by monitoring the optical density. The structure of the liposome and the reconstitution of CD40 were confirmed by cryo-TEM. The results of the present study show that the detergent ratio had an effect on the structure of the liposome and the amount of CD40 that was reconstituted into the liposome.     

A single step protein assay that is both detergent and reducer compatible: The cydex blue assay

Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue‐based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single‐step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta‐cyclodextrin or alpha‐cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2–10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay.     

Selected non-ionic biological detergents enhance signal intensity of intact bovine serum albumin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Recently, we showed that the signal intensity of intact protein by matrix-assisted laser desorption/ionisation (MALDI) mass spectro-metry measurement can be enhanced at least an order of magnitude by the addition of Tween80 to the analyte solution. We did not ascertain whether this effect was limited to Tween80 or if it was more universal of biological detergents. This paper discusses our investigations into this question. A variety of chemically diverse detergents were added to analyte solutions containing bovine serum albumin (BSA) to determine whether there was significant signal enhancement. The addition of Tween20, Tween80, Triton X100 and Triton X-114 improved the attainable sensitivity of intact protein MALDI mass spectrometry compared to spectra acquired without detergent. In some cases there was considerable improvement in signal--for example, with Triton X-100 two charge states (the +1 and +2) of BSA (3.9 fmol) could easily be observed. Another advantage of this process is that the detergent can be added directly to the matrix solution reducing sample handling and preparation time. We propose this phenomenon results from the ability of these detergents to increase the solubility of the protein via hydrophobic and hydrophilic interactions between the detergent and protein. The increased solubility allows for more uniform deposition of the analyte/-matrix mixtures producing an evenly distributed layer of analyte especially useful for data acquisition using an automated laser firing sequence.     

Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study

Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure–property relationships important for future design of novel amphiphiles.

Newly developed amphiphiles, designated ACAs, are not only efficient at extracting G protein-coupled receptors from the membranes, but also conferred enhanced stability to the receptors compared to the gold standards (DDM and LMNG).     

Dispersion of Carbon Nanotubes with “Green” Detergents

Solubilization of carbon nanotubes (CNTs) is a fundamental technique for the use of CNTs and their conjugates as nanodevices and nanobiodevices. In this work, we demonstrate the preparation of CNT suspensions with “green” detergents made from coconuts and bamboo as fundamental research in CNT nanotechnology. Single-walled CNTs (SWNTs) with a few carboxylic acid groups (3–5%) and pristine multi-walled CNTs (MWNTs) were mixed in each detergent solution and sonicated with a bath-type sonicator. The prepared suspensions were characterized using absorbance spectroscopy, scanning electron microscopy, and Raman spectroscopy. Among the eight combinations of CNTs and detergents (two types of CNTs and four detergents, including sodium dodecyl sulfate (SDS) as the standard), SWNTs/MWNTs were well dispersed in all combinations except the combination of the MWNTs and the bamboo detergent. The stability of the suspensions prepared with coconut detergents was better than that prepared with SDS. Because the efficiency of the bamboo detergents against the MWNTs differed significantly from that against the SWNTs, the natural detergent might be useful for separating CNTs. Our results revealed that the use of the “green” detergents had the advantage of dispersing CNTs as well as SDS.     

Foldable Detergents for Membrane Protein Study: Importance of Detergent Core Flexibility in Protein Stabilization

Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM−Hs) and 1,2-ethylenediamine (TZM−Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM−Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM−Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM−Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.     

Detergent Mediated Effects on the High-Performance Liquid Chromatography of Proteins

Abstract

Present chromatographic systems for the high-performance liquid chromatography (HPLC) of hydrophobic-proteins are generally limited to size-exclusion or ion-exchange chromatography. A major stumbling block to the successful chromatography of membrane-proteins is their limited solubility. Detergent is usally required to solublize these proteins. This detergent causes some problems in size-exclusion chromatography, but does not always interfere with the separation. It is more deleterious in anion-exchange chromatography, where ionic detergents can poison the column, and reversed-phase chromatography, where strong interactions can occur between the stationary phase and detergent. Successful chromatography of membrane-proteins requires favorable detergent/stationary-phase interactions that enhance, rather than interfere with, the separation.

To study these “detergent-mediated effects” a series of protein standards were chromatographed by reversed-phase HPLC. The column was then saturated with detergent and the standards rechromatographed. To evaluate any irreversible effects (caused by detergent/stationary-phase interactions) the column was washed extensively and re-evaluated. Following this procedure a variety of stationary-phases and detergents were tested.

The results of these studies showed that resolution was enhanced by detergent. Retention time was generally uneffected, but peak width was noticeably decreassed. Proteins were separated by fast gradients and recovered in high yields (95–99%). A C-18 stationary-phase gave better resolution than a C-8 stationary-phase. In all cases studied the column was irreversibly modified.

A final test of the “detergent-modified” columns was the chromatography of membrane-proteins. Prior attempts at the reversed-phase HPLC of these proteins had resulted in either no sample recovery, or of very low yields of purified protein. An acetylcholinesterase containing sample chromatographed as series of fused peaks, two of which were found to contain cholinesterase activity. Human lymophocyte function-antigen chromatographed as a single peak and was recoved with a 95% yield.     

Two-Dimensional Detergent Expansion Strategy for Membrane Protein Studies

Detergents are the most frequently applied reagents in membrane protein (MP) studies. The limited diversity of one-head-one-tailed traditional detergents, however, is far from sufficient for structurally distinct MPs. Expansion of detergent repertoire has a continuous momentum. In line with the speculation that detergent pre-assembly exerts superiority, herein we report for the first time cross-conjugation of two series of monomeric detergents for constructing a two-dimensional library of dimeric detergents. Optimum detergents stood out with unique preferences in the systematic evaluation of individual MPs. Furthermore, unprecedented hybrid detergents 14M8G and 14M9G enabled high-quality EM study of transporter MsbA and NMR study of G protein-coupled receptor A_2AAR, respectively. Given the abundance of cross-coupling chemistries, comprehensive diversity could be readily covered that would facilitate the finding of new detergents for the manipulation of thorny MPs and innovation of the functional and structural study in future.     

Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.     

Detection of protein from detergent solutions by probe electrospray ionization mass spectrometry (PESI-MS)

Detergents are necessarily used for different extraction protocols of proteins from biological cells or tissues. After the extraction, elimination of detergent is necessary for the better performance of electrospray ionization mass spectrometry (ESI-MS). Elimination of detergents is laborious and time-consuming, and also sample loss may be unavoidable. Probe electrospray ionization (PESI) developed in our laboratory has been found to be tolerant to the presence of salts and buffers in sample solutions. In this report, it was examined whether PESI is applicable to the sample solutions that contain high-concentration of detergents. It was found that PESI is highly tolerant to the presence of sodium dodecyl sulphate, cetyl trimethylamminium bromide, Triton X100 and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate compared with conventional ESI and nanoESI. Therefore, PESI can be a potential analytical tool for direct analysis of protein extracts and digests containing high-concentration detergents.     

Use of surface plasmon resonance to study the adsorption of detergents on poly(dimethylsiloxane) surfaces

This paper demonstrates the use of surface plasmon resonance to study adsorption (either reversible or irreversible) of detergents on PDMS surfaces in real time. The surface plasmon resonance measurements can directly provide information about the adsorption/desorption processes of detergents on the surface revealing the durability of the adsorbed layer and the anticipated degree of the EOF. Hydroxypropyl methylcellulose very strongly adsorbs onto PDMS and can be considered both a semipermanent layer and stable semipermanent coating. Adsorbed SDS or CTAB layers were stable for several minutes upon rinsing the surface with solution not containing the detergent. It was shown that SDS coated onto PDMS in microchips has the potential to afford similar separations in PDMS as found in conventional fused silica capillaries.     

Sodium-aluminium-silicates in the washing process Part X. Cobuilders and optical brighteners

This study is concerned with the physico-chemical background to the substitution of phosphates in laundry detergents, with special emphasis on combinations of zeolite A with other detergent ingredients, especially water-soluble cobuilders and optical brighteners. The most important action mechanisms of zeolite A / cobuilder systems were investigated for a variety of washing conditions. It is shown that the builder performance of zeolite A is often reinforced by small amounts of water-soluble substances such as polycarboxylates. These detergents are able to compete, in terms of performance, with products containing a high proportion of phosphate. Further, the interaction of zeolite A with fluorescent whitening agents in detergent powders was studied. It is shown that the photophysical properties of a typical optical brightener are negatively influenced by ionic builders and surfactants. In contrast to sodium triphosphate, zeolite A can enhance the appearance of powders even in the presence of anionic surfactants.Lecture presented at the International Symposium on Zeolites, Zeolites as Catalysts, Sorbents and Detergent Builders, Würzburg, September 4–8, 1988.     

Selective Extraction and Quantitation of Polyoxyethylene Detergents and its Application in Protein Determination

Abstract

A simple, rapid and selective method for the nearly quantitative extraction of high amounts (10% w/v) of polyoxyethylene nonionic detergents like Triton X-100^R is presented based on solvent extraction with 1, 2-dichloroethane. Protein determination after extraction of protein containing solutions can be accomplished using the Bradford assay¹ or other standard methods. High protein recoveries can be obtained. The detergent analysis by HPLC using reversed-phases is not disturbed in the presence of proteins and therefore provides a fast and elegant method for the exact quantitation of nonionic detergents.