Stability of rhodopsin in detergent solutions.

The thermal stability of lipid-free rhodopsin in solutions of a homologous series of alkyltrimethylammonium bromide detergents and one nonionic detergent, dodecyl-beta-maltoside, has been studied as a function of detergent concentration. Rhodopsin thermal stability increases with increasing chain length within the homologous series of ionic detergents, and for chain lengths greater than 10 carbon atoms increases with increasing detergent concentration up to a "critical" concentration that depends on the chain length. Stability also increases with increasing detergent concentration for rhodopsin in solutions of the nonionic detergent. These results may be rationalized in terms of the dependence of micelle packing density on the detergent chain length, head group, and concentration.     

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.     

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.     

Effect of detergents on the association of the glycophorin a transmembrane helix

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.     

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide‐binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full‐length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.     

Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent

We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.     

Destabilization of collagen structure by amides and detergents in solution

The effects of amides and detergents on collagen to gelatin transition have been studied at neutral pH. Simple amides denature the protein. The substitution of H-atoms by the alkyl groups at the nonpolar end of amide increases the effectiveness of the compounds in destabilizing the collagen structure whereas substitution of the H-atom at the polar amide end shows marginal effects on the collagen transition. The capabilities of these reagents to denature collagen are much less pronounced than their effects on denaturing globular proteins. Anionic detergents are found to destabilize collagen at very low concentrations (below their cmc values). In this respect, the effects of the detergents on collagen are comparable to the denaturing effects of the detergents on globular proteins. The effect of detergents increases with the increase in the length of the alkyl chain. The structure of the anion in the detergent is also important as seen from the lower potency of the sulfonate containing detergent compared to the sulfate containing detergent in denaturing collagen. Cationic and nonionic detergents do not denature collagen.     

BIOLOGICAL EFFECTS OF SURFACTANTS. V. GROWTH AND ANTHOCYANIN PRODUCTION BY CALLUS CULTURES OF DIMORPHOTHECA

Effects of five homologous series of amphoteric, anionic, and nonionic surfactants on growth and anthocyanin production by callus cultures of Dimorphotheca sinuata were examined. The phytotoxicity of amphoteric sulfobetaines increased with alkyl chain length and reached a plateau at 14 to 16 carbon atoms. In the case of the anionic sodium alkyl sulfates, the dodecyl derivative caused the greatest inhibition. Higher molecular weight ionic detergents were less toxic, probably due to their high Krafft temperatures and concomitant reduced solubility. The nonionic higher alcohol ethoxylates were generally less inhibitory than the ionic detergents and had little or no effect below their critical micelle concentrations. Fresh weights of tissues and anthocyanin production by Dimorphotheca callus cultures declined with increasing surfactant concentrations.     

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.     

The effect of detergents on firefly luciferase reactions

The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity. Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.     

Protein-cationic detergent interaction. Interaction of bovine serum albumin and other proteins with alkylpyridinium bromides studied by viscosity, gel filtration and spin-label methods.

Viscosity, gel filtration and spin-labelling methods have been used to study the influence of alkylpyridinium bromides on the conformation of bovine serum albumin and other proteins. Cationic detergents cause partial unfolding of the native protein molecules. The magnitude of these changes increases with increasing length of the detergent hydrocarbon chain. When cationic detergents are added to reduced and carboxymethylated bovine serum albumin the observed changes are opposite to those found in native protein.     

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.     

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.     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions

Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of beta-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55 degrees C at neutral pH and below pH 4.5 at 20 degrees C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, beta-octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH less than 3.5, small, reversible changes in protein structure became evident. The thermal stability of porin is also increased by beta-octyl glucoside as evidenced by a transition temperature 15-20 degrees C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of beta-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca2+, Mg2+) to the experimental system did not improve the thermal reversibility.     

Molecular organization of pheophytin a in aqueous solutions of detergents

Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge.