Detergent Sodium Dodecyl Sulfate Research Articles

Permeability barrier of the Gram-negative bacterial outer membrane with special reference to nisin

The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1- N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to 1 mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.

The virulence regulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic Vibrio species.

The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution of V. cholerae as an intestinal pathogen.

High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels.

We recently described a method for recovering polyacrylamide-gel-separated bacterial lipopolysaccharides (LPS) based on the sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 microm average size gel microparticles into water. With this procedure, the recovery of rough- or semismooth-type LPS after 3 h elution is about 70-80%, while that of smooth LPS is only about 10%. Here we evaluated whether a simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration-dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K-235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS.

Interactions between HIV-1 gp41 Core and Detergents and Their Implications for Membrane Fusion

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.

Solubility of Excited States in Micelles: The Nucleophilic Aromatic Photosubstitution of 3-Nitrophenyl Ethers in Anionic Micellar Solution

The alkaline photohydrolysis of a series of n-alkyl 3-nitrophenyl ethers was investigated in aqueous micellar solutions of the anionic detergent sodium dodecyl sulfate (SDS). Although SDS micelles strongly inhibit the OH- mediated photohydrolysis of the hydrophobic n-decyl ether, they have very little effect on the photohydrolysis of the corresponding n-butyl ether and actually increase the quantum yield for photohydrolysis of the ethyl and methyl ethers. This unexpected behavior is shown to be the consequence of a rather large decrease in the micellar solubility of the 3-nitrophenyl ether chromophore upon excitation to the triplet state, coupled with a micellar effect on the decomposition of the intermediate sigma complex. On the basis of recent linear solvation free energy relationship analyses of solute incorporation into SDS micelles, this change in micellar solubility is attributed to an enhancement of the hydrogen bond basicity in the excited triplet state of the 3-nitrophenyl ether chromophore.

Poly(vinyl chloride)-based macrocyclic membrane sensors for magnesium

Poly(vinyl chloride)-based membranes of macrocycles 4,11-dimethyl-2,4,9,11-tetraethyl-1,5,8,12-tetraaza cyclotetradeca-1,8-diene (I) and 4,11-dioxa-2,9-dimethyl-1,5,8,12-tetraaza cyclotetradeca-1,8-diene (II) with sodium tetraphenyl borate (STB) as an anion excluder and dibutyl phthalate (DBP), dioctyl phthalate (DOP), dibutylbutyl phosphonate (DBBP) and 1-chloronaphthalene (CN) as plasticizing solvent mediators were prepared and investigated as magnesium selective electrodes. The best performance was observed having the composition (II)–PVC–STB–DBP in the ratio 2:10:1:7, which works well over a wide concentration range (1.9×10 −6 to 1.0×10 −1 M) with a Nernstian slope of 29 mV per decade of activity between pH 2.5 and 6.5. These electrodes have been found to be chemically inert showing a fast response time of 15 s and were used over a period of 3 months with good reproducibility ( S=±0.2 mV). The selectivity coefficient values for mono-, di- and trivalent cations indicate excellent selectivity for Mg 2+ over a large number of cations. The electrodes have also been used successfully in partially non-aqueous medium and as an indicator electrode in the potentiometric titration of Mg 2+ with EDTA. Anions such as Cl − and SO 4 2− do not interfere in the working of the electrode. The practical utility of the membrane sensor has also been observed in solutions contaminated with detergents (cetyltrimethyl ammonium bromide and sodium dodecyl sulphate). Above all, the membrane sensor has been very successfully used to analyse some babyfood products and soft drinks for the determination of Mg 2+.

Activation of Human Neutrophil NADPH Oxidase by Phosphatidic Acid or Diacylglycerol in a Cell-free System: ACTIVITY OF DIACYLGLYCEROL IS DEPENDENT ON ITS CONVERSION TO PHOSPHATIDIC ACID

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis.

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.

Infection-related surface proteins on conidia of the nematophagous fungus Drechmeria coniospora

The adhesion of conidia of Drechmeria coniospora to the nematode Panagrellus redivivus was reduced after treatment of the conidia with Pronase E, or the detergents sodium dodecyl sulphate (SDS) and dodecyltrimethylammonium bromide (DTAB) suggesting involvement of proteinaceous compounds in the adhesion process. In the TEM the thick extracellular pad covering the adhesive bud of the conidia was completely removed after treatment with Pronase E. After treatment with SDS or DTAB the proteinaceous compounds appeared to be dissolved leaving mainly carbohydrates in the pad as observed on OsO4 without and OsO4 with Ruthenium red-stained material, respectively. The detergent extracts after SDS and DTAB treatments contained nine and seven peptides, respectively, with molecular masses in the range from 6 to 80 kDa on SDS-PAGE gels, and five biotinylated peptides were found in each extract, after blotting to nitrocellulose membranes, indicating that these were surface proteins. None of the detergent extracts was able to reduce adhesion of the conidia after treatment of the nematodes. The detergent extracts contained protease- and phosphatase activity. The protease inhibitor, chymostatin, inhibited infection of nematodes and growth of the conidia, suggesting the involvement of chymotrypsin-like proteases in the infection process. On gelatin-containing substrate gel electrophoresis two proteases were clearly chymostatin sensitive.

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1.

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5-10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90 degrees C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85min at 100 degrees C and 12 min at 110 degrees C.

Protein Purification for the Lowry Assay: Acid Precipitation of Proteins in the Presence of Sodium Dodecyl Sulfate and Other Biological Detergents

The Lowry protein assay is widely used to quantify proteins in the laboratory. As the assay is susceptible various interfering substances, the protein test sample is frequently first purified by precipitating the proteins using trichloroacetic acid (TCA). However, TCA precipitates proteins poorly in the presence of sodium dodecyl sulfate (SDS), a detergent very commonly used in biological research. The inhibitory effect of up to 1% SDS on protein precipitation is effectively overcome by a combination of 5% TCA and 1% phosphotungstic acid (PTA). The TCA/PTA mixture also precipitates proteins readily in the presence of other detergents such as 0.1% Tween 20, Triton X-100 or Nonidet P-40. TCA/PTA has the capacity to precipitate a wider cross-section of proteins than TCA alone, the combination of acids remaining effective in the presence of SDS and other detergents.

Chemical/Physical Retting of Flax Using Detergent and Oxalic Acid at High pH

An oxalic acid-based system for chemically retting flax is tested and compared with enzymatic retting. The retting effect is substantially higher at pH 10 and 11 than at lower pH levels, and the optimal oxalic acid concentration is 50 mmol. The presence of the strong detergent sodium dodecyl sulfate (SDS) further enhances retting. The rate of retting with the oxalic acid/SDS mixture at pH 10 is strongly temperature dependent, and at 75°C the process is completed within 2 hours. Tests of fiber properties ( e.g., tex, tenacity, and elongation) indicate that the chemical/physical retting method pro duces fibers of textile quality that can be spun into flax-rich blends with cotton.

Identification of two Shigella flexneri chromosomal loci involved in intercellular spreading.

The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.

Submicellar bile salts stimulate phosphatidylcholine transfer activity of sterol carrier protein 2

Abstract To explore a potential role for sterol carrier protein 2 (SCP2, also known as non-specific lipid transfer protein) in hepatocellular phospholipid trafficking, we examined the influence of submicellar bile salt concentrations on phosphatidylcholine (PC) transfer activity of SCP2. We measured rate constants for first-order transfer of sn-1 palmitoyl, sn-2 parinaroyl PC, a naturally fluorescent self-quenching phospholipid between model membranes. Purified bovine liver SCP2 promoted transfer of PC from donor to acceptor small unilamellar vesicles. Taurine- and glycine-conjugated bile salts (anionic steroid detergent-like molecules), at concentrations well below their critical micellar concentrations, stimulated PC transfer activity of SCP2 80- to 140-fold. Rate constants increased in proportion to bile salt concentration, temperature, and bile salt–membrane binding affinity. Sodium taurofusidate, a conjugated fungal bile salt analog, also activated PC transfer whereas no effect was observed with the anionic and nonionic straight chain detergents sodium dodecyl sulfate and octylglucoside, respectively. Thermodynamic and kinetic analyses of PC transfer support a mechanism in which bile salts stimulate SCP2 activity by partitioning into donor vesicles and enhancing membrane association of SCP2. These results imply that under physiological conditions, SCP2 may contribute to hepatocellular selection and transport of biliary PCs.—Leonard, A. N., and D. E. Cohen. Submicellar bile salts stimulate phosphatidylcholine transfer activity of sterol carrier protein 2. J. Lipid Res. 1998. 39: 1981–1988.

Multicellular growth of bacteria

Pseudomonas aeruginosa is an opportunistic pathogen that can colonize catheters and the lungs of cystic fibrosis patients. It is protected from antimicrobial agents by the ability to form biofilms. LasI encodes the production of the quorum sensing signal N-(3-oxododecanoyl)-l-homoserine lactone. Davies et al.[1xThe involvement of cell-to-cell signals in the development of a bacterial biofilm. Davies, D.G. et al. Science. 1998; 280: 295–298Crossref | PubMed | Scopus (1978)See all References][1]now report that a lasI mutant of P. aeruginosa is defective in normal biofilm formation. This mutant formed a biofilm that was only 20% as thick as the one formed by wild-type P. aeruginosa and was sensitive to the detergent sodium dodecyl sulfate. Clearly, these results have important medical implications and point towards new strategies for the control of biofilm-forming pathogens. However, they also provide an example of an increasing trend pointing to complex multicellular traits in bacteria. The idea of multicellularity among `simple' bacteria has been around for many years and met with early skepticism. It is now clear that a bacterial colony holds many exciting mysteries worthy of exploration.

Differential Effects of Detergents on Keratinocyte Gene Expression

We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on epidermal keratinocytes is qualitatively different from the other detergents tested, which might have implications for in vitro toxicology studies that use cell biologic parameters as a read-out. We would hypothesize that detergents cause skin injury by several mechanisms that include direct cellular toxicity, disruption of barrier function, and detergent specific effects on cellular differentiation, as demonstrated here for sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, and sodium laurate.

Extraction of DNA from mycoplasmas.

The manipulation of genetic material for the purpose of diagnosis or analysis almost always requires the preparation of sample to expose genomic nucleic acid or the extraction and purification of DNA. Molecular techniques, such as restriction enzyme analysis, Southern hybridization, random amplified polymorphic DNA (RAPD) analysis, and nucleic acid sequencing, rely on the purity and integrity of DNA for consistent results. Generally, cells are disrupted mechanically or by detersive agents, proteins inactivated by heat denaturation or enzymatic digestion, and cellular material removed from nucleic acids by phase separation in solvents. Many other methods have been described for the preparation of DNA suitable for amplification and restriction enzyme analysis. Some include new reagents that are mixtures of solvents and result in the reduction of handling time, whereas others are special polymers that sequester or chelate materials other than nucleic acids. The method that combines the action of the detergent sodium dodecyl sulfate (SDS), the proteolytic enzyme proteinase K (PK), and the solvent phenol is widely used for DNA extraction from a variety of eukaryotic and prokaryotic sources. With the exception of pulse-field gel electrophoresis (PFGE), which requires unsheared DNA, this method provides DNA suitable for most techniques, including PCR.

Quantitative Evaluation of Neurotensin Receptor Purification by Immobilized Metal Affinity Chromatography

Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success. Strong binding to the metal resin is essential for purification when expression levels are low. We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed inEscherichia coliat a level of about 0.1% of membrane protein. Receptors with six C-terminal histidine residues did not bind to nickel resin in the presence of the anionic detergent sodium dodecyl sulfate. In contrast, partial purification assessed by densitometry of Coomassie-stained gels was achieved using the nonionic detergents dodecyl maltoside or Triton X-100 (53% pure), or a detergent mixture containing the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (46% pure). Linking a highly charged epitope tag to the histidine tail did not affect the nickel-binding properties of receptors. The level of purification was substantially improved (72% pure) by extending the histidine tail to 10 residues because this allowed stringent washes at high imidazole concentration to remove nonspecifically bound contaminants. This strategy not only resulted in efficient purification of receptors from crude membranes, but also worked particularly well for single-step purification from total cell lysates, resulting in 340-fold purification of functional neurotensin receptor.

Using Surface Plasmon Resonance Spectroscopy To Measure the Association of Detergents with Self-Assembled Monolayers of Hexadecanethiolate on Gold

This paper describes the use of surface plasmon resonance (SPR) spectroscopy to measure the rates and extents of association of four detergentssodium dodecyl sulfate (SDS), β-octyl glucoside, Triton X-100, and Tween 20to self-assembled monolayers (SAMs) of alkanethiolates on gold. SAMs presenting hexaethylene glycol groups resisted the adsorption of all four detergents. These same detergents associated with hydrophobic SAMs presenting methyl groups; the concentration of detergent molecules on the surface was 120−280 pmol/cm2. The associations of the detergents with the hydrophobic SAM were described well by the Langmuir adsorption isotherm. The dissociation constants Kd (M) for the desorption of the detergents from the surface correlated with the critical micelle concentration (cmc, M) of the detergents in solution, and followed the relationship cmc ≈ 7 (±2)Kd. The efficacy of SDS in removing the protein fibrinogen adsorbed on a hydrophobic SAM depended strongly on the concentration of detergent. SDS at a...

Using quantum chemistry to predict solubilization in detergent micelles

Experimental K s values for the solubilization of 66 non-ionic solutes in micelles of the anionic detergent sodium dodecyl sulfate can be reproduced using quantum chemical surrogate parameters, the calculation of which requires only a knowledge of the solute molecular structure. The parameters evaluated include the solute molar volume ( V x ), the solute HOMO and LUMO energies, the magnitudes of the charges of the most positive hydrogen and most negative atom of the molecule ( q + and ¦q −¦ , respectively), the molecular dipole moment and the solute “dipolarity” (the molecular polarizability divided by V x ). The resultant linear solvation free energy relationship (LSER) based on these surrogate parameters ( log K s = −0.487 + 1.27q + −3.76¦q −¦ + 2.97V x ) is chemically reasonable and consistent with the LSER for micellar solubilization based on experimentally-derived solute parameters. The use of quantum chemical parameters to develop LSERs should thus prove to be a convenient approach for predicting solubilities in organized media from solute molecular structure.