PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

Interaction of beef liver lipase with mixed micelles of tripalmitin and Triton X-100

When concentrated dispersions of tripalmitin in Triton X-100 are added to reaction mixtures containing soluble beef liver lipase, the rate of hydrolysis of tripalmitin increases with incubation time. When the diluted substrate is aged at 37 degrees C for 3 hr before the addition of enzyme, the rate of hydrolysis is greater than the rate with freshly diluted dispersions and is constant for at least 2 hr. The reciprocal of the rate of hydrolysis is a complex function of the reciprocal of the substrate concentration when measured with freshly diluted substrate dispersions. A linear relationship between these reciprocals is obtained when measured with aged preparations of substrate. The rate and extent of increase of the velocity of hydrolysis of the aged substrate in relation to the velocity of hydrolysis of freshly diluted substrate are directly proportional to the substrate concentration and inversely proportional to the Triton X-100 concentration. The apparent V(max) of beef liver lipase for tripalmitin in diluted and aged dispersions is independent of the Triton X-100 concentration, while the apparent K(m) is inversely proportional to the Triton X-100 concentration. The apparent K(m) for tripalmitin complexes at zero Triton X-100 concentration was judged to be 7.5 x 10(-5) m. The molecular size of dispersion complexes does not change significantly as dispersions are aged. The spherical diameter of the complexes assessed by gel filtration techniques is in the order of 100 A.     

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.     

Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.     

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Radioiodination of nonionic detergents of the alkyl-phenol class: Triton X-100

A method is described by which nonionic detergents of the alkyl-phenol class can be labeled with ¹²⁵I. The detergent is labeled to a high specific activity, which provides a sensitive method for the detection of detergent molecules bound to proteins, and, in addition, may provide a method by which proteins may be labeled indirectly.     

Laser light-scattering characterization of mitochondrial complex III-Triton X-100-phospholipid mixed micelles

Bovine-heart mitochondrial complex III was purified in the presence of Triton X-100, and the size and shape of the resulting protein-surfactant-phospholipid mixed micelles were investigated by laser light-scattering. The protein appears to be present in the form of a dimer, irrespective of temperature (between 25 and 40 degrees C) and protein concentration (between 0.5 and 5 mg/ml). The molecular weight of the micelle increases with temperature from 600 000 (25 degrees C) to 692 000 (40 degrees C). The variation of the solvent second virial coefficient in this temperature range suggests that, with increasing temperature, some of the free surfactant molecules become integrated in the mixed micelles. The average quadratic radius of gyration of these is of 42 +/- 5 nm, corresponding in our case to an ellipsoidal shape.     

Proton magnetic resonance relaxation studies on the structure of mixed micelles of Triton X-100 and dimyristoylphosphatidylcholine.

Proton magnetic resonance and gel chromatographic studies on mixtures of phospholipid and the nonionic surfactant Triton X-200 have shown that at temperatures above the thermotropic phase transition of the phospholipid and below the cloud point of Triton, mixed micelles are present at molar ratios above about 2:1 Triton/phospholipid. Proton T1 and T2 (from line widths) relaxation times are reported for protons in Triton micelles and in mixed micelles of Triton and dimyristoylphosphatidylcholine at a molar ratio of 3:1 Triton/phospholipid. The T1 values and their temperature dependence and the activation energies of the various Triton proton groups appear to reflect internal motions of the Triton molecules in the micelle. Measurements of the T1/T2 ratio and frequency dependence (55-220 MHz) suggest that the hydrophobic tert-butyl group in Triton is observed under extreme narrowing conditions. The T1 and T2 values of Triton are unchanged in the presence of phosphatidylcholine. The T1 values of various protons of dimyristoylphosphatidylcholine in mixed micelles are similar to those reported for the phospholipid in sonicated vesicles, which are used as membrane models, and presumably the same coupled trans-gauche motions dominate. The T2 values for the terminal methyl and choline methyl protons in the phospholipid are longer than those reported for these groups in vesicles. Hence, the motion of the phospholipid in the mixed micelles appears to be less restricted than in vesicles. T1 measurements in H20/D20 mixtures are consistent with the idea that water does not penetrate the hydrophobic core of the mixed micelles, while water does solvate the polar oxyethylene and choline methyl groups. Titration with Mn2+ confirms that the oxyethylene and choline methyl groups are on the exterior of the mixed micelle while the hydrophobic groups are located in the micellar interior.     

Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

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.     

Biodegradation of octylphenol polyethoxylate surfactant Triton X-100 by selected microorganisms

Octylphenol polyethoxylate (OPEO(n)) surfactants are used in numerous commercial and industrial products. Large amounts of such surfactants and their various residual biodegradation by-products are ultimately released into the environment. OPEO(n) biodegradation was performed in this study using pure cultures of Pseudomonas species and strains under different environmental conditions. Environmental factors including the pH, nitrogen sources, and growth kinetics of the cells were investigated. The intermediates of Triton X-100 biotransformation were detected by high performance liquid chromatography-mass spectrophotograph (HPLC-MS). We found the highest specific growth rate (mu) was 0.56 h(-1) and this was achieved by strain E with an initial concentration of Triton X-100 of 5000 mg L(-1). A pH level of 7 was most favorable for cell growth for all five strains. The highest specific growth rate was achieved using (NH(4))(2)SO(4) as the sole nitrogen source for strain E. Strain A showed an enhancement of growth when between 0.2 and 1.4 mg L(-1) of H(2)O(2) was added. Detection of intermediates was possible after four days of transformation and the octylphenol triethoxylate (OPEO(3)) peak was predominant, while the high molecular weight peaks had all disappeared. The kinetic analysis demonstrated that the greatest maximum specific growth rate (mu(max)) and the greatest saturation constant (K(s)) of 0.83 h(-1) and 5.24 mg L(-1), respectively, were obtained for strain E in 5000 mg L(-1) Triton X-100. The higher K(i) revealed that strain A was resistant to higher Triton X-100 concentrations.     

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.     

Regulation of yeast phosphatidylserine synthase and phosphatidylinositol synthase activities by phospholipids in Triton X-100/phospholipid mixed micelles

The regulation of purified yeast membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) and phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) activities by phospholipids was examined using Triton X-100/phospholipid mixed micelles. Phosphatidate, phosphatidylcholine, and phosphatidylinositol stimulated phosphatidylserine synthase activity, whereas cardiolipin and the neutral lipid diacylglycerol inhibited enzyme activity. Phosphatidate was a potent activator of phosphatidylserine synthase activity with an apparent activation constant (0.033 mol %) 88-fold lower than the apparent Km (2.9 mol %) for the surface concentration of CDP-diacylglycerol. Phosphatidate caused an increase in the apparent Vmax and a decrease in the apparent Km for the enzyme with respect to the surface concentration of CDP-diacylglycerol. Phosphatidylcholine and phosphatidylinositol caused an increase in the apparent Vmax for phosphatidylserine synthase with respect to CDP-diacylglycerol with apparent activation constants of 3.4 and 3.2 mol %, respectively. Cardiolipin and diacylglycerol were competitive inhibitors of phosphatidylserine synthase activity with respect to CDP-diacylglycerol. The apparent Ki value for cardiolipin (0.7 mol %) was 4-fold lower than the apparent Km for CDP-diacylglycerol, whereas the apparent Ki for diacylglycerol (7 mol %) was 2.4-fold higher than the apparent Km for CDP-diacylglycerol. Phosphatidylethanolamine and phosphatidylglycerol did not affect phosphatidylserine synthase activity. Phosphatidylinositol synthase activity was not significantly effected by lipids. The role of lipid activators and inhibitors on phosphatidylserine synthase activity is discussed in relation to overall lipid metabolism.     

A Triton X-100-hydroxyapatite procedure for a rapid purification of bovine heart cytochrome-c oxidase. Characterization of the cytochrome-c oxidase/Triton X-100/phospholipid mixed micelles by laser light scattering

Cytochrome-c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) has been isolated from bovine heart mitochondria by the combined use of the non-ionic detergent Triton X-100 as solubilizing agent and hydroxyapatite chromatography as the most important step in the purification of this membrane protein. This method is fast and very reproducible. The enzymic complex, purified in the form of protein/detergent/phospholipid mixed micelles, contains 9.7 mumol heme a per mg protein, and has a high molecular activity (500 mol cytochrome c per s per mol enzyme). These mixed micelles have been studied by laser light scattering, which has shown that the average molecular weight of the micelles is 540 +/- 80 kDa. This implies that cytochrome-c oxidase is purified in the form of a dimer. The average quadratic radius of gyration of the micelles is 40 +/- 10 nm, corresponding in our case to an approximately spherical shape.     

Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.     

Structural studies on apolipoprotein B: controllable heterogeneity of the complex formed with the surfactant, Triton X-100

Apolipoprotein B complexed with Triton X-100 (T-ApoB) has been isolated from human low density lipoprotein (LDL). Preparations are heterogeneous when analyzed by sedimentation velocity, with a major 12 S species and minor 17 S species present. The 12 S T-ApoB complex possesses a molecular weight of 880,000 containing 400,000 daltons of protein. Hydrodynamic measurements on this complex are consistent with a prolate ellipsoid model having an axial ratio of 13:1 and 0.22 g/g of bound water. Heterogeneity results from the irreversible aggregation of 12 S complexes into discrete 17 S and faster sedimenting components. A significant finding is that three determinants of this T-apoB heterogeneity could be elucidated and controlled. First, the initial state of aggregation is mainly influenced by the technique by which Triton and LDL are mixed. Second, once isolated, T-ApoB complexes slowly but spontaneously undergo further aggregation at 4 degrees C; the rate and extent of aggregation is enhanced remarkably with increasing temperature. Finally, reagents that unfold and expose protein structure (perchlorate, thiocyanate, and reducing reagents) lead to increased aggregation. The ability to control heterogeneity carries important implications for other studies concerning interactions of apoB with surfactants and lipids.