Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.     

High water solubility and fold in amphipols of proteins with large hydrophobic regions: oleosins and caleosin from seed lipid bodies

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.     

Long chain arginine esters: a new class of cationic detergents for preparation of hydrophobic ion-paired complexes.

The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent-like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration-dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA.     

Photopolymerized phospholipid vesicles Stability and retention of hydrophilic and hydrophobic marker substances

Multilamellar vesicles were formed from photopolymerizable analogs of phosphatidylcholine. The polymerized vesicles differed markedly from conventional vesicles in terms of their stability to mechanical and chemical perturbations. Thus, polymerized vesicles, but not conventional ones, retained their overall physical integrity subsequent to ultrasonication or to exposure to organic solvents or detergents. Treatment of photopolymerized vesicles with detergents such as sodium dodecyl sulfate caused the release of entrapped hydrophilic solutes; however, lipophilic solutes were retained by the polymerized vesicles under circumstances in which conventional vesicles were completely solubilized. Thus, photopolymerized phospholipid membranes represent hybrid entities which seem to blend some of the characteristics of conventional lipid bilayers with properties more typical of polymer membranes.     

A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.     

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents

The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.     

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.     

Perturbation of the aminoacyl-tRNA synthetase complex by salts and detergents. Importance of hydrophobic interactions and possible involvement of lipids

In order to gain some insight into the structural parameters important for aminoacyl-tRNA synthetase complex formation, we have examined the effect of various salts and detergents on the stability and structure of the synthetase complex. Certain neutral salts were found to inactivate aminoacyl-tRNA synthetase activities in the complex, and the order of effectiveness in this process followed a classical Hofmeister series. In addition, one of these salts, NaSCN, was also effective in partially dissociating the complex. Detergents varied in their ability to inactivate synthetases, with ionic detergents being most effective and nonionic detergents being much less destructive. Detergents, by themselves, could partially disrupt the complex; however, in the presence of 1 M NaCl, nonionic detergents did lead to considerable dissociation of synthetases and generation of low molecular weight forms of these enzymes. Removal of lipids from the complex with the nonionic detergent, Triton X-114, rendered arginyl-tRNA synthetase sensitive to the addition of NaCl. However, this salt sensitivity was abolished by readdition of a lipid extract isolated from the complex. These results implicate hydrophobic interactions in the stability of the synthetase complex, and suggest the possible involvement of lipids in maintaining its structural integrity.     

Purification of membrane proteins in SDS and subsequent renaturation

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.     

Two-dimensional electrophoresis of soybean root plasma membrane proteins solubilized by SDS and other detergents

Proteins solubilized from enriched soybean root plasma membrane with sodium dodecyl sulphate (SDS) and selected non-denaturing detergents (octyl-β-d-glucopyranoside, Zwittergent 312, Zwittergent 314, Zonyl FSK, and Nonidet P-40) were electrophoresed in two-dimensions by standard procedures. The basic electrophoretogram ‘fingerprint’ was similar for all detergents tested. However, differences in the total number of polypeptides resolved and the presence or absence of certain polypeptides on specific two-dimensional gels indicated some selectivity. Of all detergents tested, SDS solubilized the most polypeptides (ca 95) and provided the best resolution. The other detergents solubilized 50–80 polypeptides with varying resolution. Of those tested, octyl-β-d-glucopyranoside consistently provided the best balance between the number of polypeptides resolved (ca 70) and the level of resolution. The results suggest that selected detergents may prove useful in plant plasma membrane studies which require non-denaturing conditions.     

Enhanced solubilization of immunoreactive proteins from Brugia malayi adult parasites using cetyltrimethylammonium bromide

To determine an optimal method for extracting immunoreactive proteins from filarial parasites, we have subjected Brugia malayi adult worms to a variety of solubilization regimens and compared the results. The parasites were extracted in one of seven detergents (including anionic, cationic, nonionic, and zwitterionic compounds) under varying conditions of pH, detergent concentrations, and incubation time. The individual antigen preparations were then compared both by one-dimensional SDS-PAGE and by immunoblotting analysis using a serum pool from individuals resident in an area endemic for lymphatic filariasis. The cationic detergent cetyltrimethylammonium bromide (CTAB) at 1.0% concentration, pH 7.2, consistently solubilized more proteins immunoreactive with the sera tested. Additionally, CTAB never failed to solubilize immunoreactive proteins solubilized by those other detergents or combinations of detergents studied.     

Properties of native calf spleen NAD+ glycohydrolase immobilized by hydrophobic interaction

NAD+ glycohydrolase, an amphipathic membrane-bound enzyme, solubilized from calf spleen microsomes with detergents and purified, was immobilized by hydrophobic interactions on octyl-Sepharose. Conditions are described for optimal adsorption on the gel. The immobilized enzyme remained catalytically active (hydrolase and transglycosidase activities) and could be used to prepare nicotinic acid analogs of NAD(P)+.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Solubilization conditions for hydrophobic membrane protein,oleosin, in soybeans

Oleosins are amphipathic proteins found in soybeans that give stability to oil-bodies by surrounding them. Furthermore, oleosins have broad biotechnological applications in protein purification and in immobilization as a tagging parther. In this study we provide the proper combinations of detergents and chaotropes for solubilization of oleosins. The solubilization effects of each detergent were compared by the results of SDS-PAGE analysis, from the density measurement of each band using an image analysis program. Among the twelve chemicals tested, 3-(decyldimethylammonio) propanesulfonale inner salt (SB 3–10), nonaethylene glycol octylphenyl ether (NP-40), and n-dodecyl-β-d-maltoside (DM) were determined to be effective for oleosin solubilization. In addition, urea, thiourea, and reducing agents played roles in enhancing the effects of the detergents. The best results were obtained with 2.5% of each detergent in combination with 4 M urea and 2 M thiourea at room temperature. The zwitterionic detergent, SB 3–10, is recommended as the best non-denaturant for the solubilization of soybean oleosins with the advantage of its easy removal by dialysis due to its low molecular weight and a high critical micelle concentration.     

Lipopeptide detergents designed for the structural study of membrane proteins

The structural study of membrane proteins requires detergents that can effectively mimic lipid bilayers, and the choice of detergent is often a compromise between detergents that promote protein stability and detergents that form small micelles. We describe lipopeptide detergents (LPDs), a new class of amphiphile consisting of a peptide scaffold that supports two alkyl chains, one anchored to each end of an alpha-helix. The goal was to design a molecule that could self-assemble into a cylindrical micelle with a rigid outer hydrophilic shell surrounding an inner lipidic core. Consistent with this design, LPDs self-assemble into small micelles, can disperse phospholipid membranes, and are gentle, nondenaturing detergents that preserve the structure of the membrane proteins in solution for extended periods of time. The LPD design allows for a membrane-like packing of the alkyl chains in the core of the molecular assemblies, possibly explaining their superior properties relative to traditional detergents in stabilizing membrane protein structures.     

An evaluation of the hydrophobic interactions of chick muscle acetylcholinesterase by charge shift electrophoresis and gradient centrifugation

The hydrophobic interactions of globular forms of acetylcholinesterase from adult and embryonic chick muscles have been analyzed by sucrose gradient centrifugation and non denaturing polyacrylamide gel electrophoresis. The presence of positively- or negatively-charged detergents influences the electrophoretic migrations of hydrophobic globular forms, whereas the mobility of hydrophilic components is unchanged. We defined an hydrophobicity index (HI) which quantitatively reflects this interaction.Globular forms of acetylcholinesterase were isolated in preparative sucrose gradients of muscle extracts. The G₁ form (5 S) appeared as a single band in electrophoresis, the G₂ form (7 S) under two and the G₄ form (11 S) under three electromorphs. The G₁ and the G₂ forms interacted with detergents: this resulted in a shift in their sedimentation in sucrose gradients upon removal of detergents, and in a modification of their electrophoretic migrations in the presence of charged detergents (HI = 1.0 for G₁, HI = 1.7 for G₂). The G₄ form was heterogenous: one band (G_4f) did not interact with detergent (HI = 0.1). The other variants (G_4i and G_4s) were clearly hydrophobic (HI = 0.5 and HI = I respectively). The hydrophilic and hydrophobic variants of the G₄ form however, were not resolved by sedimentation analysis performed in the presence of Triton X100, but their separation was improved in the presence of 10-oleyl-ether. Therefore, the combination of electrophoretic and sedimentation methods, as described in this paper, can be used successfully for subdividing a single molecular form (size isomer defined by hydrodynamic parameters) into several constituents differing by their hydrophobic interactions.     

On choosing a detergent for solution NMR studies of membrane proteins

Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10^-7cm²/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Negative staining of integral membrane proteins

This review describes aspects of negative staining of isolated integral membrane proteins. Detergents play a central role in the isolation of membrane proteins and also in their solubility in aqueous solutions. Specimens of mixed micelles of membrane proteins and nonionic detergents can be easily prepared as long as the detergent concentration remains above the critical micellar concentration. Membrane proteins involved in the process of photosynthesis have been taken as examples to illustrate their interaction with different detergents. Upon negative staining, mixed micelles of membrane proteins and detergents show characteristic top and side view projections. On their sides, mixed micelles can easily aggregate into strings.     

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.