Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

Liquid scintillation counting of aqueous solutions of potassium salts in a Triton X-100/toluene scintillant

The use of a toluene/Triton X-100 scintillant for counting ¹⁴C in aqueous solutions of potassium salts including potassium hydroxide has been investigated. Suitable conditions for the counting of CO₂ entrapped in potassium hydroxide are described. Quench correction by automatic external standard channels ratio procedures has been found to be of value with optimised conditions of counting.     

Improved technique for accurate and convenient assay of biological reactions liberatingCO2

Use of a convenient and inexpensive apparatus for trapping¹⁴CO₂ from biological reactions in small volumes is described. Alternative scintillants for estimating¹⁴CO₂ trapped in KOH are compared, toluene methoxyethanol (2:1) based scintillants and Instagel (TM-Packard) were efficient and more stable than the dioxane/Cab-O-Sil or toluene/Triton X-100 mixtures usually used. The usefulness of the technique is illustrated with microassays of OMP decarboxylase (EC 4.1.1.23), OMP pyrophosphorylase (EC 2.4.2.10) in the fission yeastSchizosaccharomyces pombe.     

A water-miscible, nonhazardous liquid scintillation cocktail

The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts ¹⁴C with 80% efficiency and ³H with 17% efficiency. It has a high flash point (over 300°F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate

Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the d-malate production activity, until the maximal malease activity was reached. Incubation of P. pseudoalcaligenes cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating that cell lysis had occurred. The rate at which the d-malate production activity increased was dependent on the Triton X-100 concentration and on the cell density. Also the rate at which lysis occurred depended on the Triton X-100 concentration.     

Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

The Non-Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H+-ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H⁺-ATPase of plants of Mesembryanthemum crystallinum performing C₃ photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H⁺-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K_M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C₃ and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H⁺-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H⁺-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H⁺-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H⁺-ATPase is important for enzyme structure and function.     

Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein

Protein has been selectively extracted from isolated chicken erythrocyte nuclear envelope by (1) dilute MgCl₂/Triton X-100 followed by (2) concentrated MgCl₂/Triton X-100 solutions. Certain proteins appear to be selectively dissolved in the first solvent and may occur in the nuclear envelope primarily as lipoproteins. Among the proteins insoluble in the low MgCl₂/Triton X-100 wash, as well as in 500 mM MgCl₂ without Triton previously used in the preparation of the envelope fraction, the quantitatively major polypeptides dissolve in a combination of high MgCl₂ and Triton X-100. Further, much of this dissolved protein precipitates when the MgCl₂ concentration is lowered by dialysis. The insolubility of these proteins appears to result from a combination of ionic and hydrophobic interactions and may explain the resistance of nuclei to various manipulative procedures including nonionic detergent washes. The procedures described provide a route for gently and selectively dissolving representative proteins from the nuclear envelope lipoprotein matrix and from the envelope “residual” protein.     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

The effects of cations on the structure and photochemistry of the photosystem II core complex

We have studied the effects of cations and detergents on the structure (molecular weight) and photochemistry of Triton X-100 Photosystem II subchloroplast particles (TSF-IIa). The effect of Mg²⁺ ions on activity depended on the Triton X-100 content of the preparation. If the residual Triton X-100 was not removed prior to assay, MgCl₂ increased the rate of electron transport, acting at a site on the reducing side of Photosystem II. Lowering the pH also increased the rate of electron transport. If the Triton X-100 was removed from the particles, both MgCl₂ and NaCl caused a decrease in the rate of electron transport. Addition of Triton X-100 caused a reversible decrease in the number of active Photosystem II reaction centers. Both cations and Triton X-100 had a profound effect on the molecular weight of the Photosystem II particles as determined by gel filtration. At 20 °C, addition of 0.05% Triton X-100 decreased the molecular weight from a high value (≥800,000) to 250,000. At 4 °C, addition of 1 mm MgCl₂ or 100 mm NaCl increased the molecular weight of the complex. In the absence of these salts 67% of the protein eluted with a molecular weight of 460,000 (the rest was >800,000-in the void volume). In the presence of these salts all of the material had a molecular weight of ≥800,000. A similar effect was observed when the pH was lowered from 8 to 6. Further work is needed to determine whether there is a correlation between the changes in molecular weight and activity.     

The use of a triton X-100-based scintillant for counting fractions from density gradients

The properties of an aqueous scintillation mixture containing butyl-PBD as the sole scintillant and using Triton X-100 as emulsifier are described. This counting mixture, which is considerably cheaper than other published mixtures for aqueous samples, is shown to perform extremely satisfactorily with polysomes and RNA labeled by prior injection of [¹⁴C]orotic acid. When, however, this counting mixture is used with ³H-labeled samples, the density gradient solutes sucrose and cesium chloride are shown to quench the counting of RNA and polysomes but not of toluene or orotic acid.     

Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate((R)) has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6log after 30min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

Impact of Triton X-100 on alpha 2-antiplasmin (SERPINF2) activity in solvent/detergent-treated plasma

Large-pool solvent/detergent (SD) plasma for transfusion exhibits reduced alpha 2-antiplasmin (alpha2-AP; SERPINF2) functional activity. The reason for the loss of alpha2-AP has not been described and could be due to the SD incubation itself and/or to the processing steps implemented to remove the solvent and the detergent. We have studied alpha2-AP activity during six down-scale preparations of plasma virally-inactivated by 1% (v/v) TnBP combined with two different non-ionic detergents, either 1% Triton X-100 or 1% Triton X-45, at 31 degrees C for 4h. The SD-treated plasmas were then extracted with 7.5% (v/v) soybean oil, centrifuged at 3800 x g for 30 min, and subjected to hydrophobic interaction chromatography (HIC) to remove the SD agents. Control runs without TnBP and Triton were performed to evidence possible impacts of each process step on alpha2-AP activity. TnBP, Triton X-100, and Triton X-45 were measured at all stages of the processes to evaluate potential interferences with the alpha2-AP assay. Alpha 2-AP activity was about 10% that of starting plasma after 1% TnBP-1% Triton X-100 incubation and about 50% after oil extractions, centrifugation, and HIC. By contrast about 73% of the antiplasmin activity was found after the incubation with 1% TnBP and 1% Triton X-45, 88% after removal of the SD agents by oil extractions, 90% after centrifugation and 92% after HIC. The control runs performed without SD agents showed that the process steps did not affect the alpha2-AP activity. In conclusion, the agent altering alpha2-AP activity in SD-plasma is Triton X-100. The choice of detergents for the SD viral inactivation of therapeutic plasma fractions used in patients at risk of fibrinolysis should consider the impact on alpha2-AP activity.     

Immunologic comparison of the conformations of apolipoprotein B. Investigation of methodologies for the reconstitution of delipidated and denatured apolipoprotein B with nonionic surfactants

Immunologic probes have been used to examine the conformation of apolipoprotein B (apo-B) as it exists within native low density lipoprotein (LDL) after lipid displacement with Triton X-100 and after denaturation with guanidine hydrochloride organic solvent delipidation and reconstitution with Triton X-100. Antigenic expression was assayed in two systems: by using either Triton X-100 or bovine serum albumin to maintain protein solubility. Apo-B delipidated by lipid displacement using Triton X-100 was virtually identical to LDL-apo-B in both systems, as assayed by polyclonal antisera prepared in rabbits against either antigen. Thus the native antigenic sites are preserved, although the displacement of the lipid core of LDL drastically alters the physical properties of the particle. Apo-B delipidated by solvent extraction in guanidine was reconstituted with Triton X-100 by several methods, and the products were examined immunologically. One method yielded a product that resembled apo-B as delipidated with Triton X-100, although full reconstitution could not be achieved. Nevertheless, Triton promoted refolding of apo-B to reform partial native structure as judged immunologically. By using both physical and immunologic methods for assessing structure, it is clearly evident that the perceptions of the conformational states of reconstituted apo-B can be very different, and multiple criteria need to be used to assess lipoprotein reconstitution.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models

The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X-100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X-100 demembranated sperm while 0.01% and 0.05% Triton X-100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1% Triton X-100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X-100 permeabilized sperm models. Reactivated motility of 0.05% Triton X-100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.     

Increase in Striatal [3H]Muscimol Binding Following Intrastriatal Injection of Kainic Acid: A Denervation Supersensitivity Phenomenon

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of [³H]muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound [³H]muscimol was significantly increased at the injected site, whereas no significant alteration of [³H]muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (K_D) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (B_max) therein. This significant increase in [³H]muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X-100, a non-ionic detergent. No statistically significant difference in amounts of [³H]muscimol binding was detected when the preparations from the KA-treated and non-treated CS were preincubated with 0.05% Triton X-100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X-100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X-100, while the total number of binding sites (B_max) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X-100 in vitro may increase the amount of specifically bound [³H]muscimol to CS preparations by a similar molecular mechanism.