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PubMed2005

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.

Anions↗

PubMed1987

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane.

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.

Anions↗

PubMed2004

Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins.

BACKGROUND: Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. METHODS: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. RESULTS: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach. CONCLUSIONS: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.

Animals↗

PubMed2000

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.

4-Chloro-7-nitrobenzofurazan↗

PubMed1993

Detergent interaction with band 3, a model polytopic membrane protein.

The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2005

Less skin irritation from alcohol-based disinfectant than from detergent used for hand disinfection.

BACKGROUND: The benefit of alcohol-based disinfectant used on normal skin has been debated. OBJECTIVES: The objective of the present study was to compare the effects of repeated exposure to an alcohol-based disinfectant, a detergent and an alcohol-based disinfectant/detergent alternately for 10 days, including noninvasive measurements in the evaluation. Skin reactivity in irritated skin after a 4-week interval was also evaluated. MATERIALS AND METHODS: Detergent, disinfectant and alternate disinfectant and detergent were applied twice daily every 10 min for 1 h to the ventral upper arms and forearms of 17 healthy volunteers. A control area was included. After 4 weeks an SLS patch was applied to each area. Irritant reactions were quantified with a visual score recording and measurements of transepidermal water loss (TEWL) and skin colour were performed on days 1, 5, 11, 38 and 40. RESULTS: On day 5 the detergent caused a higher visual score than either disinfectant applied alone or alternate disinfectant and detergent, P < 0.05. On day 11 detergent and alternate disinfectant and detergent caused a higher score than disinfectant, P < 0.05. An increased irritant response for detergent compared with disinfectant alone and alternate disinfectant and detergent was confirmed by TEWL evaluations on days 5 and 11, P < 0.001, while no significant difference was found by colour measurement. After 4 weeks, when evaluated by colour measurement, significantly less response to the SLS patch was found on the disinfectant-treated area compared with the control area and detergent area, and a similar trend was found for TEWL, although it was not statistically significant. CONCLUSION: Alcohol-based disinfectant caused less visible skin irritation and less skin barrier disruption than the use of detergent. The alternate use of detergent and disinfectant caused less irritation than the use of detergent, and a possible interaction between the two irritants was not indicated. After 4 weeks there was a tendency for decreased skin reactivity in the skin areas which had 4 weeks previously been exposed to disinfectant.

Adult↗

PubMed1996

The association of different detergents with the photosynthetic reaction center protein of Rhodobacter sphaeroides R26 and the effects on its photochemistry.

Detergent-free reaction centers from Rhodobacter sphaeroides R26 were used to study the solubilization of reaction centers in various detergents and their effects on reaction center photochemistry. 500 +/- 100 n-octyl-beta-D-glucopyranoside or 51 +/- 5 Triton X-100 molecules were associated with one reaction center. For N.N-alkylamine N-oxide detergents with chain lengths in the range from 8-13 carbon atoms, the number of detergent molecules associated with the reaction centers increased with decreasing chain length. The amount of detergent molecules associated with the reaction centers decreased almost tenfold if the pH was increased from pH 6 to pH 10. The addition of 5% 1,2,3-heptanetriol to various detergents lowered the detergent/reaction center ratio by a factor of two compared to that for the pure detergent. The detergent concentration at which solubilization of the reaction center occurs was close to the critical micelle concentration for all detergents studied. The absorption band at 865 nm of the primary donor in the reaction center shifts to 846 nm when detergent was removed from the reaction center; upon resolubilization with various detergents, this band shifts back to 865 nm. In 80-90% of the detergent-free reaction centers, the secondary electron transfer from QA to QB was inhibited: this electron transfer was restored after re-addition of detergent.

Cytochrome c Group↗

PubMed1997

Growth hormone induces detergent insolubility of GH receptors in IM-9 cells.

In this study, we examined human growth hormone (hGH)-induced changes in nonionic detergent solubility characteristics of its receptor (hGHR). Exposure of IM-9 cells to hGH caused a time- and concentration-dependent loss of immunoblottable detergent-extractable hGHRs and a corresponding accumulation of receptors in a detergent-insoluble pool. At 37 degrees C, the loss of detergent-soluble and the accumulation of detergent-insoluble hGHRs both preceded hGH-induced loss of total cell hGHRs. The detergent-insoluble receptor pool was progressively enriched in an apparent disulfide-linked form of the hGHR. Exposure to hGH at 4 degrees C allowed hGH-induced hGHR disulfide linkage but did not promote changes in receptor detergent solubility, indicating that hGHR detergent insolubility cannot be explained solely by the formation of that linkage. Experiments carried out with hGH at 20 degrees C and with the phorbol ester, phorbol-12,13-myristate acetate, at 37 degrees C indicated that loss of detergent-soluble hGHRs can be uncoupled from accumulation of detergent-insoluble receptors. From these data, we envision at least two related, but separable, trafficking pathways taken by hGHRs after their surface interaction with hGH:1) ligand-mediated endocytosis and degradation (accounting for only some of the receptors lost from the detergent-soluble fraction) and 2) ligand-mediated accumulation in a detergent-insoluble subcellular fraction (arising largely from receptors redistributed from the detergent-soluble fraction).

Cell Line↗

PubMed2005

Detergent-resistant, ceramide-enriched domains in sphingomyelin/ceramide bilayers.

When cell membranes are treated with Triton X-100 or other detergents at 4 degrees C, a nonsolubilized fraction can often be recovered, the "detergent-resistant membranes", that is not found when detergent treatment takes place at 37 degrees C. Detergent-resistant membranes may be related in some cases to membrane "rafts". However, several basic aspects of the formation of detergent-resistant membranes are poorly understood. To answer some of the relevant questions, a simple bilayer composition that would mimic detergent-resistant membranes was required. The screening of multiple lipid compositions has shown that the binary mixture egg sphingomyelin/egg ceramide (SM/Cer) exhibits the required detergent resistance. In detergent-free membranes composed of different mixtures of SM and Cer (5-30 mol % of Cer) differential scanning calorimetry, fluorescence spectroscopy, and fluorescence microscopy experiments reveal the presence of discrete, Cer-enriched gel domains in a broad temperature range. In particular, at temperatures below SM phase transition ( approximately 40 degrees C) two gel (respectively Cer-rich and SM-rich) phases are directly observed using fluorescence microscopy. Although pure SM membranes are fully solubilized by Triton X-100 at room temperature, 5 mol % Cer is also enough to induce detergent resistance, even with a large detergent excess and lengthy equilibration times. Short-chain Cers do not give rise to detergent resistance. SM/Cer mixtures containing up to 30 mol % Cer become fully soluble at approximately 50 degrees C, i.e., well above the gel-fluid transition temperature of SM. The combined results of temperature-dependent solubilization and differential scanning calorimetry reveal that SM-rich domains are preferentially solubilized over the Cer-rich ones as soon as the former melt (i.e., at approximately 40 degrees C). As a consequence, at temperatures allowing only partial solubilization, the nonsolubilized residue is enriched in Cer with respect to the original bilayer composition. Fluorescence microscopy of giant unilamellar vesicles at room temperature clearly shows that SM-rich domains are preferentially solubilized over the Cer-rich ones and that the latter become more rigid and extensive as a consequence of the detergent effects. These observations may be relevant to the phenomena of sphingomyelinase-dependent signaling, generation of "raft platforms", and detergent-resistant cell membranes.

Animals↗

PubMedDate not supplied

Strategies toward maximum detergent efficiency.

Detergent Z demonstrated the most reliable cleaning and detergent delivery system. After the initial detergent evaluation, the data were compared against all detergents. It was found that acceptably cleaned items ranged from 37% to 85% per evaluation. Labor and material costs could be reduced greatly with the use of detergent Z, which had the best cleaning results. We also observed the advantage of a sensor in the detergent delivery system which can override some less obvious equipment malfunctions. Since detergent Z also exhibited the least amount of residue build-up, a savings in maintenance costs could be expected later on. The vendor of detergent Z showed great interest and concern for our department's needs and quality assurance by offering us numerous technical resources during the evaluation. One year following our detergent studies, detergent Z and its related line of cleaning products are being utilized in our hospital. We found that the four months spent analyzing detergents used in our utensil washers provided an interesting resource project that has been a positive investment in improved operations for the decontamination area. In Central Service alone, 61.7 hours a week were saved by the change in products. Quality of cleaning continues to be high. The practice of a weekly acid bath for the utensil washers has been discontinued; the acid bath will now be given when necessary, and has not been done since detergent Z was put into use six months ago. During the next fiscal year, savings of detergent, utilities and labor are conservatively projected at $30,000.

Central Supply, Hospital↗

PubMedDate not supplied

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.

Adenosine Triphosphate↗

PubMed1995

Phospholipid solubilization during detergent extraction of rhodopsin from photoreceptor disk membranes.

The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23 degrees C were ineffectual at 4 degrees C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin-lipid-detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein-lipid-detergent mixed micelles was also consistently richer in PC than that present in lipid-detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein-lipid-detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related detergents.

Animals↗

PubMed1976

The action of a binary nonionic detergent on a kidney membrane fraction.

The disruption of a kidney cortex microsomal membrane preparation by a binary, nonionic detergent, was followed by using as markers, the changes in total protein content, and (Na+, K+)-ATPase in a supernatant fraction. Both markers responded similarly to changes in pH, microsome concentration and detergent concentration, but responded differently for time-dependent studies. The (Na+, K+)-ATPase activity was increased 2.2-fold (76.1 mumoles Pi/mg protein/h, 95% ouabain-sensitive) by a single detergent treatment and 3.5-fold (92% ouabain-sensitive) by a sequential detergent treatment. Changes in the critical micelle concentration (cmc) were observed for varying detergent and protein concentrations, which suggest interactions of monomeric detergent with the membrane. The peak of (Na+, K+)-ATPase activity occurred above the cmc which suggests the participation of micelles in releasing the enzyme from the membranes. Hill plots of the protein released as the detergent concentration was varied showed a change in the slope near the cmc indicating a four-fold increase in the binding of detergent to membranes as the detergent concentration is increased above the cmc. These results suggest that the disruption of membranes by detergent involves the binding of detergent monomers to the membrane followed by the formation of co-micelles of the detergent with segments of the membrane to complete the separation process.

Adenosine Triphosphatases↗

PubMed2006

Women's strategies for handling household detergents.

Performing household-cleaning chores involves the use of chemical detergents; these are commonly believed to provide cleaner and safer households. Occupational health studies have associated health risks with detergents, including respiratory and skin problems. Women are the major users of household detergents, as they are in general the primary homemakers. Detergent-handling strategies including storage, use, and precautionary measures affect women's exposure to chemical detergents and thus affect their health. Studies investigating the behavioral component of chemical exposure to detergents at the domestic level are scarce. In this study of detergent-handling strategies, women's potential exposure was investigated by an ergonomics methodology combining in-depth interviews and observations. The participants were 28 women who were responsible for cleaning chores in their households. The women lived in Nabaa, a low socioeconomic, religiously and ethnically diverse neighborhood on the outskirts of Beirut, Lebanon. Information on how women in Nabaa used and perceived household detergent, added to the understanding of their potential chemical exposure. All the study subjects washed dishes, cleaned bathrooms, and swept floors on at least a daily basis. We found that more than half of the participants stored different incompatible detergents in close proximity to each other. Many of the women stated that they over-consumed detergents. A large proportion mixed different detergents together but most of them opened the windows while cleaning for ventilation. The majority did not wear gloves and did not follow label directions. The participants were consistently exposed to chemical detergents and minimal precautionary measures were taken. Cleanliness and hygiene had a strong cultural value and were of utmost importance to women who prioritized them over excessive exposure to chemicals. The use of an ergonomics methodology provided access to important information concerning how women make choices that affect their environmental exposure.

Adult↗

PubMed2003

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.

Binding Sites↗

PubMed1993

Transitional steps in the solubilization of protein-containing membranes and liposomes by nonionic detergent.

Membrane solubilization by dodecyl maltoside was studied, using Ca(2+)-ATPase membranes and liposome preparations as prototypes of biological membranes. In equilibrium dialysis experiments, transition from saturable incorporation of monomeric detergent into the membrane to cooperative binding already occurred at a free detergent concentration about 50% of the cmc. This transition was discontinuous for unilamellar liposomes of dioleoylphosphatidylcholine, but gradual for Ca(2+)-ATPase membranes and multilayered liposomes of sarcoplasmic reticulum lipid. Equilibrium detergent binding by Ca(2+)-ATPase membranes (expressed on the basis of lipid content) was the same as for detergent binding by multilamellar liposomes of sarcoplasmic reticulum lipid. Equilibration involving cooperative binding was considerably delayed (for many days) if detergent was presented gradually to the membranous preparations in nonmicellar form by diffusion across the dialysis membrane, while equilibration of detergent occurred rapidly if detergent in micellar form was added directly to the membrane preparations. In contrast, equilibration was rapid in both directions if detergent was added at levels below that required to initiate cooperative binding. Detergent interaction resulted in a biphasic decrease in light scattering of Ca(2+)-ATPase membranes. The first of these decreases coincided with the onset of cooperative binding, while the second one was associated with a decreased sedimentability during ultracentrifugation, i.e., with usual criteria of solubilization. The concentration at which this occurred corresponded to the level of free detergent at which lipid, after detergent solubilization, segregated from detergent after gel chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1978

Interaction of an amine oxide detergent with lecithin vesicles as studied by nuclear magnetic resonance.

The interaction of an amine oxide detergent with single bilayer lecithin vesicles was investigated with proton and phosphorus magnetic resonance. The addition of the detergent micelles to vesicles suspensions leads to rapid detergent incorporation into the vesicle bilayer, resulting in a heterogenous vesicle population. Initially, some vesicles take up the equivalent of one detergent micelle, whereas others contain no detergent. Subsequently, the detergent is distributed between the vesicles by vesicle-vesicle collisions. This can be followed by the change in the Pr3+-shifted spectral positions of the detergent and lecithin head groups with time. From the intensity of the head-group signals, it can be concluded that after about 20 h the detergent is almost equally distributed between the outer and inner vesicle membrane monolayers. Vesicles obtained by cosonication of the detergent and lecithin take up metal ions. This ion permeability depends on the vesicle concentration and can be attributed to vesicle-vesicle or vesicle-mixed micelle collisions. Egg lecithin vesicles are stable against the detergent up to molar ratios of detergent to lecithin of 0.2--0.3. At larger ratios mixed micells and multibilayers are formed. Measurements of proton spin-lattice relaxation times confirmed that the internal architecture of the vesicle bilayer is almost unaffected by the incorporated detergent.

Detergents↗

PubMed1997

Pathway of detergent-mediated and peptide ligand-mediated refolding of heterodimeric class II major histocompatibility complex (MHC) molecules.

We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101. Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.

Circular Dichroism↗