The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Structural changes induced by Triton X-100 on sonicated phosphatidylcholine liposomes

Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.     

Effect of Triton X-100 on the physical properties of liposomes

The interaction of the nonionic detergent Triton X-100 with phospholipid bilayers of liposomes made of egg yolk phosphatidylcholine was studied through the behavior of several physical properties. The dielectric permittivity spectra between 30 kHz and 13 MHz, the viscosity, the density, and the d.c. conductivity (1 kHz) of aqueous liposomes suspensions at various mole ratios were measured at 22 degrees C. For detergent-to-phospholipid ratios lower than 3, a dielectric relaxation process of characteristic frequency of about 50 kHz was recorded. This process does not appear for the liposomes in water, and becomes smaller and smaller for detergent-to-phospholipid ratios higher than 3. The viscosity of these suspensions showed a biphasic behavior, being remarkably increased by the detergent for concentration ratios lower than 3. The measured d.c. conductivity of these samples showed no relation with this process, being slightly increased when the detergent content is increased. As a conclusion of these results a well defined concentration range appears where the phospholipid organization changes forming highly asymmetrical structures.     

The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.Abbreviations DEAE diethylaminoethyl - MeOH methanol - SDS sodium dodecyl sulphate     

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 degrees C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 degrees C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures "kinetic traps" may occur not only at 4 degrees C but at higher temperatures as well (e.g. 37 degrees C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 degrees C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization (Dt(sol)) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 degrees C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 degrees C. In this system, we observe a bell-shaped dependence of Dt(sol) on temperature, with a minimum at 37 degrees C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 degrees C.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Effects of Triton X-100 concentration and incubation temperature on carboxyfluorescein release from multilamellar liposomes

Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence ‘end point’ for distearoyl-l-α-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22°C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72°C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100.

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X-100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane-surfactant interactions.     

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: A P-NMR study

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by ³¹P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.     

Studies on Triton X-100 detergent micelles.

Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.     

Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles

A detailed kinetic analysis of purified yeast membrane-associated phosphatidate phosphatase was performed using Triton X-100/phosphatidate mixed micelles. Enzyme activity was dependent on the bulk and surface concentrations of phosphatidate. These results were consistent with the "surface dilution" kinetic scheme (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) where phosphatidate phosphatase binds to the mixed micelle surface before binding to its substrate and catalysis occurs. Phosphatidate phosphatase was shown to physically associate with Triton X-100 micelles in the absence of phosphatidate, however, the enzyme was more tightly associated with micelles when its substrate was present. The enzyme had 5- to 6-fold greater affinity (reflected in the dissociation constant nKsA/chi) for Triton X-100 micelles containing dioleoyl-phosphatidate and dipalmitoyl-phosphatidate when compared to micelles containing dicaproyl-phosphatidate. The Vmax for dioleoyl-phosphatidate was 3.8-fold higher than the Vmax for dipalmitoyl-phosphatidate, whereas the interfacial Michaelis constant chi KmB for dipalmitoyl-phosphatidate was 3-fold lower than the chi KmB for dioleoyl-phosphatidate. The specificity constants (Vmax/chi KmB) of both substrates were similar which indicated that dioleoyl-phosphatidate and dipalmitoyl-phosphatidate were equally good substrates. Based on catalytic constants (Vmax and chi KmB), dicaproyl-phosphatidate was the best substrate with an 11- and 14-fold greater specificity constant when compared to dioleoyl-phosphatidate and dipalmitoyl-phosphatidate, respectively.     

Characteristics of phospholipase D in reverse micelles of Triton X-100 and phosphatidylcholine in diethyl ether

Summary Phospholipase D, from cabbage, is active in reverse micelles formed from its substrate phosphatidylcholine and Triton X-100 in diethyl ether. The activity is optimum at w₀=12.5. The increase of the molar ratio of Triton X-100/substrate from 1:4 to 2:1 results in an activity decrease by 25 %. At 136 mM Triton X-100 the K_M value in reverse micelles is 136 mM, whereas it is 0.40 mM in the aqueous system containing SDS.     

Dependence of yeast permeabilization with Triton X-100 on the cell age

Summary Acid phosphatase activity and protein release were determined in cell suspensions ofYarrowia lipolytica andTorulospora delbrueckii at different stages of their growth by permeabilization with Triton X-100. The effect of the surfactant on the cell permeability did not depend on the cell age.     

Triton X-100 reacts with chlorophyll in the presence of chlorophyllase

Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) catalyses the transesterification of chlorophylls with the surfactant Triton X-100, which is widely used in the preparation and study of this enzyme. The preparation and some properties of water-soluble tritonyl chlorophyllide esters are described. A mechanism for the role of Triton X-100 as an inhibitor in chlorophyllase-catalyzed hydrolysis and transesterification of chlorophylls is proposed. Bacteriochlorophyl a also has been employed as a substrate for green plant chlorophyllase.