Interaction Of Triton X-100 Research Articles

Electrosteric stabilization of colloidal TiO2 nanoparticles with DNA and polyethylene glycol for selective enhancement of UV detection sensitivity in capillary electrophoresis analysis.

A new approach to selectively enhance the ultraviolet (UV) detection sensitivity of titania (TiO2), albeit in the presence of silica (SiO2), alumina (Al2O3), and zinc oxide (ZnO), nanoparticles in capillary electrophoresis (CE) analysis was developed. Interactions of Triton X-100 (TX-100), polyethylene glycol (PEG),and deoxyribonucleic acid (DNA) with TiO2 nanoparticles produced larger CE-UV peaks at various enhancement factors. Single-stranded DNA (ssDNA) was a more effective adsorbate than double-stranded DNA (dsDNA) due to its flexible molecular structure that participated in a stronger interaction with TiO2 nanoparticles via its sugar-phosphate backbone. Disaggregation of TiO2 nanoparticles upon DNA binding due to electrosteric stabilization was validated using dynamic light scattering. PEG coating of TiO2-DNA nanoparticles further enhanced the UV detection sensitivity in CE analysis by providing extra electrosteric stabilization. This analytical technique, which involves binding of TiO2 nanoparticles with DNA followed by coating with PEG, has allowed us to achieve progressively an enhancement factor up to 13.0 ± 3.0 - fold in analytical sensitivity for the accurate determination of disaggregated TiO2 nanoparticles. Graphical Abstract Selective enhancement of UV detection sensitivity for TiO2 nanoparticles via electrosteric stabilization using ssDNA and PEG.

Interactions of dipeptides with Triton X-100 in aqueous solution: A volumetric and spectroscopic study

The interactions of dipeptides with Triton X-100 in aqueous solution have been investigated by means of density, fluorescence spectroscopy and UV–vis spectroscopy. The standard partial molar volume (V2,ϕo), standard partial molar volume of transfer for dipeptide from water to aqueous Triton X-100 solution (ΔtVo) and partial molar expansibility (Eϕo) have been calculated from density data. Fluorescence spectroscopy was used to estimate the critical micellar concentration (ccmc) and micelle aggregation number of Triton X-100 in aqueous dipeptide solutions. Effects of temperature and hydrocarbon chain length of dipeptides on the volumetric properties of dipeptide and critical micelle concentration (ccmc) of Triton X-100 were examined. The pyrene fluorescence spectra were also used to study the change of micropolarity produced by the interactions of Triton X-100 with dipeptides. From the results of UV–vis absorption spectra, the binding constant between dipeptide and Triton X-100 above the ccmc was determined. The results have been interpreted in terms of solute–solvent interactions and structural changes in the mixed solutions.

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: Influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 °C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air–water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.

Interaction of non-ionic surfactants with hepatic CYP in Prochilodus scrofa

Cytochromes P450 (CYP) constitute a superfamily of hemeproteins that play a vital role in the metabolism of a wide variety of endogenous and xenobiotic compounds. Xenobiotic metabolism and the role of CYP are of particular interest in studies regarding the prevention of the damage caused by chemical pollutants. We investigated, in this study, the interaction of Triton X-100 and Tween 80 with CYP and antioxidant defenses in Curimbatá, a Brazilian fish. Aiming to clarify the effects of non-ionic surfactants in the monooxigenase system of fish through in vitro study, the effects of Triton X-100 and Tween 80 were analyzed using monooxygenases and antioxidant system as experimental model. Total CYP and EROD were strongly inhibited by Triton X-100 and Tween 80 in a concentration-dependent way; the content of CYP was reduced until zero while EROD activity was completely inhibited in the presence of Triton X-100 and more than 40% inhibited in the presence of Tween 80. Each surfactant causes a different effect on each antioxidant enzyme. No effect was detected in SOD activity in the presence of even Triton X-100 or Tween 80. Triton X-100 increase catalase activity, while Tween 80 decreases this enzyme activity. The molecular structure of the surfactants causes the alteration of this system, since they are able to interact with the microsomal protein, especially with monooxigenase's components, altering their conformation and, consequently destroying their function. Our results suggest that surfactants can interact with components of the microsomal system leading to inhibition of CYP. Therefore, CYP activity, which has been used as a biomarker of xenobiotic exposure, should be used as a marker in association with other enzymes.

Solubilization of stratum corneum lipid liposomes by Triton X-100. Influence of the level of cholesteryl sulfate in the process

The interaction of Triton X-100 (T X-100) with stratum corneum (SC) lipid liposomes varying the proportion of cholesteryl sulfate (Chol-sulf) was investigated. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase surfactant partition coefficients ( K) were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free surfactant concentration was always similar to its critical micelle concentration (CMC) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The T X-100 ability to saturate and to solubilize SC liposomes decreased as the proportion of Chol-sulf in bilayers increased until a minimum was reached for a Chol-sulf proportion of 10%. Inversely, the surfactant partitioning into liposomes (or affinity with these bilayers) increased as the proportion of Chol-sulf increased until a maximum was reached at the same Chol-sulf proportion. Hence, when the Chol-sulf proportion in bilayers was 10% (the same that existing in the intercellular SC lipids) the ability of T X-100 molecules to interact with liposomes exhibits a minimum despite their enhanced partitioning into liposomes. These effects may be related to the reported dependencies between the level of Chol-sulf in the intercellular lipids and the abnormalities in the skin properties as the barrier function.

Alterations in stratum corneum lipid liposomes due to the action of Triton X-100: Influence of the level of ceramides on this process

The role played by the ceramides (Cer) in the interaction of Triton X-100 (T X-100) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios ( Re) and the bilayer/aqueous phase partition coefficients ( K) were determined at sublytic level by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles. Higher and lower Cer proportions than those existing in the SC (40%) led respectively to a fall and to a rise in the surfactant ability to alter these liposomes. However, the surfactant partitioning between bilayers and water (bilayer affinity with vesicles) exhibited a maximum for 40% Cer. Thus, at low Cer proportions the ability of T X-100 molecules to alter these bilayers was maximum despite their reduced partitioning into bilayers, in line with the reported interaction of the anionic surfactant model sodium dodecyl sulfate with these vesicles. These findings underline the fragility of these bilayers as an effective barrier and could explain in part the reported dependencies of low level of Cer in skin lipids and function barrier abnormalities. The fact that the free surfactant concentration needed to achieve the two interaction levels investigated was lower than the surfactant CMC indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of Cer proportion in bilayers.

Influence of the Level of Cholesteryl Sulfate Present in Stratum Corneum Lipid Liposomes on Their Stability Against Triton X-100

The interaction of Triton X-100 (TX-100) with stratum corneum (SC) lipid liposomes varying the proportion of cholesteryl sulfate (Chol-sulf) was investigated. The surfactant/lipid molar ratios and the bilayer/aqueous phase partition coefficients were determined at sublytic level by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein released from the interior of vesicles. The fact that the free surfactant concentration was always lower than the surfactant CMC indicates that permeability changes were mainly ruled by the action of surfactant monomers in all cases. The lowest surfactant ability to alter the permeability of SC liposomes and highest surfactant affinity with these bilayer structures was reached when the proportion of Chol-sulf in bilayers was 10%. Futhermore, the highest resistance of SC liposomes to be solubilized by TX-100 (via mixed micelle formation) also occurred at this Chol-sulf proportion, which corresponds to that existing in the intercellular SC lipids. These surfactant effects may be related to the reported dependencies between the level of Chol-sulf in the intercellular lipids and the abnormalities in the skin properties as the barrier function.

Interaction of Triton X-100 with cyclodextrins. A fluorescence study

The interaction of Triton X-100 (TX) with α- and β-cyclodextrins (CD) has been studied, using 2,6-p-toluidinonaphthalene sulfonate (TNS) as a fluorescent probe, by steady-state and time-resolved emission spectroscopy. The critical micellar concentration (c.m.c.) is indicated by the point of abrupt increase of emission intensity and lifetime of TNS. The apparent c.m.c. increases significantly in the presence of β-CD by as much as 28±1 times at 10 mM β-CD but remains more or less unaffected in the presence of α-CD at similar concentrations. This is attributed to the very strong binding of TX with the large β-CD cavity and negligible binding to the small α-CD. At concentrations below the c.m.c., on addition of TX to aqueous TNS solution containing β-CD the emission intensity decreases. This is ascribed to the competitive binding of TNS and TX with β-CD. This causes displacement of TNS from the CD cavity by the TX surfactant molecules. The binding constant of TX with β-CD is found to be ca. 9400±1300 L mol-1.

The interaction of Triton X-100 with purple membrane: Effect of light-dark adaptation

The effects of Triton X-100 on purple membrane have been examined for dark- and light-adapted membrane suspensions. Bacteriorhodopsin is more readily solubilized from dark-adapted than from light-adapted preparations, while light-dark adaptation does not influence phospholipid solubilization. Surfactant-induced changes in the absorption spectrum and retinal isomer distribution of bacteriorhodopsin are also illumination-dependent. These results are discussed in the light of structural data.

Interaction of Triton X-100 and octyl glucoside with liposomal membranes at sublytic and lytic concentrations. Spectroscopic studies

The molecular mechanism of the solubilisation of phospholipid bilayers by nonionic detergents was studied by turbidity changes, carboxyfluorescein fluorescence dequenching, steady-state and time-resolved fluorescence anisotropy of DPH, lifetime measurements, ANS binding and 31P-NMR. Particular attention has been paid to the effective detergent-to-lipid ratio in the lipid phase. The disturbance of the bilayer arrangement varies considerably for various detergents depending on the hydrophilic and lipophilic parts of the molecule. Small amounts of detergents with low CMC (e.g. Triton X-100) can even induce an optimisation of packing of the lipid molecules.

Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles.

Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.

Interaction of Triton X-100 with the pigment-protein complexes of photosynthetic membranes.

The interaction of the non-ionic detergent Triton X-100 with photosynthetic membrane components of Pisum sativum (pea) is described. The detergent affected both the wavelength and the intensity of the 77K fluorescence-emission peaks of both Photosystem I and Photosystem II preparations, in addition to the effects on whole thylakoids recently described by Murphy & Woodrow [(1984) Biochem. J. 224, 989-993]. Below its critical micellar concentration, Triton X-100 had no effect on 77K fluorescence emissions even after prolonged incubations of up to 30 min. Above the critical micellar concentration of about 0.16 mg X ml-1, Triton X-100 caused a dramatic increase in the intensity of the 680 nm emission. The intensity of the 680 nm fluorescence emission continued to increase as more Triton X-100 was added, until limiting concentrations of detergent were reached. These limiting concentrations were proportional to the amount of membrane present and generally occurred at Triton X-100/chlorophyll (w/w) ratios of 100-200:1. In all cases the detergent effect was seen within 10 min, and is often considerably faster, with longer detergent treatments causing no further effects. The data are discussed in terms of a three-stage mechanism for detergent solubilization of membrane components.

Detergent-like properties of polyethyleneglycols in relation to model membranes

1. Introduction 2. Materials and methods Various polyethyleneglycols (PEG) have been extensively used as chemicals inducing cell fusion ([ 1,2] and references therein). In order to obtain infor- mation on the molecular mechanisms by which PEG induces the fusion of biological membranes, various studies have been carried out on the interaction of PEG with monolayers and model membranes [l-3]. These authors conclude that PEG acts by altering the physical state of the membrane water, decreasing the surface potential and modifying the transition tem- perature (Tc) of the lipid bilayers. PEG 1000 and PEG 6000 were purchased from Merck (Darmstadt) and used without further puriflca- tion. Egg-yolk lecithin was prepared according to [7]. Aliquots of the phospholipid in chloroform (11 pmol) were evaporated to dryness and liposomes prepared as in [4,5]. The vesicle suspensions were mixed with the appropriate amounts of PEG to obtain a final phospholipid level of 1 mM, and final PEG levels of O-70% (w/v), and the various samples were left to equilibrate for 30 min at room temperature. Previous work from this laboratory [4,5] has dealt with the interaction of Triton X-100 and other deter- gents with liposomes. It was found that many soluble amphiphiles produce an increase in size of sonicated unilamellar phospholipid vesicles, giving rise to large, multilamellar liposomes, probably through a mecha- nism of ‘lysis and reassembly’ [6]. Similar detergent concentrations, when acting on non-sonlcated lipo- somes, produce only the gradual solubilization of the lipid bilayers. Turbidity measurements and electron microscopy observations were done as in [4]. Release of vesicle contents (6-carboxyfluorescein) was monitored at 25’C in a Perkin Elmer MPF3 spectrofluorimeter, according to [B]. 3. Results Considering the extensive use of PEG in cell fusion experiments, a study was undertaken in order to determine the effects of PEG on the size, permeability and solubilization of sonicated and non-sonicated phospholipid vesicles. This paper reports our results concerning PEG 1000 and PEG 6000. It is found that these reagents produce very similar effects to those caused by detergents, although at much higher con- centrations than the latter. *

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: Comparison between Mg 2+- and Mn 2+-supported enzyme activities in particulate, detergent-solubilized and detergent-insoluble fractions

Guanylate cyclase activity was determined in a 1000 g particulate fraction derived from rabbit heart homogenates using Mg 2+ or Mn 2+ as sole cation in the presence and absence of Triton X-100. With Mg 2+, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg 2+-supported enzyme activity. With Mn 2+, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg 2+ or Mn 2+, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg 2+ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg 2+-dependent activity; Mn 2+-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn 2+ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg 2+.

Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents.

The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied. Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed. Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 2.7 X 10(4) M-1. Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed. The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc. The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion. Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains. Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity. It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.

The interaction of triton X-100 with soluble proteins: Possible implications for the transport of proteins across membranes

Dodecyl sulfate complexes of two soluble proteins, serum albumin and fumarase, have been “renatured” with large excesses of the nonionic detergent Triton X-100. The resulting complexes, essentially free of dodecyl sulfate, differ in their sedimentation properties relative to the native protein and in their interaction with Triton X-100. Albumin molecules refold to a form binding only very small amounts of Triton and have a sedimentation coefficient similar to that of the non-denatured protein. On the other hand, refolded fumarase molecules have a lower sedimentation coefficient than that of the native enzyme and bind up to 1.06 mg of Triton/mg protein. It is postulated that the fumarase molecule has been turned “inside-out” by the dodecyl sulfate/Triton treatment, and the implications of such large conformational changes for protein transport across membranes are discussed.

The relations between the composition of liposomes and their interaction with triton X-100

The interaction of Triton X-100 with phospholipid bilayers of multilamellar liposomes formed from mixtures of spinal cord sphingomyelin and egg yolk lecithin in various mole ratios (all containing 1 mole of dicetylphosphate per 10 moles of phospholipid) was studied. The results indicate that the process is time dependent and is much slower than the formation of simple micelles. The time to reach the final equilibrium state is dependent on the SPM to PC mole ratio, on the Triton to phospholipid mole ratio, and on the Tirton concentration. Titration with increasing Triton concentration shows that the behavior of Triton is biphasic for all the various lipid compositions tested. For low Triton to phospholipid mole ratio there is no mass formation of mixed micelles; in addition, the Triton seems to radically increase the leakage of glucose without reducing the turbidity. This range is limited by a turning point where most of the phospholipids and about half of the Triton coprecipitate. Above this Triton to phospholipid mole ratio formation of mixed Triton-phospholipid micelles occurred followed by a drastic decline in turbidity. This turning point as well as the exact profile of the Triton effect are strongly related to the SPM:PC mole ratio. The higher the mole fraction of SPM in the membrane, the less Triton is required to reach the turning point and to cause a complete solubilization. These effects can be explained by tighter packing and stronger phospholipid-phospholipid interactions imposed by SPM and expressed as apparent microviscosity which increases upon increasing the mole fraction of SPM in the bilayer.