Removal of detergent and solvent from solvent-detergent-treated immunoglobulins.

The solvent-detergent (S/D) method was applied for inactivation of lipid-enveloped viruses during the production of immunoglobulins. Amberlite XAD-7 resin was used for removal of solvent (tri-n-butyl phosphate, TnBP) and detergent (Triton X-100) after the performed S/D inactivation procedure. The S/D reagents from the immunoglobulin preparation were adsorbed on Amberlite XAD-7, while immunoglobulins passed through the column and retained their biological activity. Using the method developed here, the final immunoglobulin preparation contains less than 1 ppm of Triton X-100 and less than 2 ppm TnBP.     

Determination of triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography

Plasma protein pools are often virus-inactivated by the solvent-detergent method, using tri-n-butyl phosphate and Triton X-100, followed by removal and determination of these compounds. We used reversed-phase high-performance liquid chromatography for the determination of Triton X-100 in coagulation factor VIII and factor IX products, Octonativ-M and Nanotiv, respectively (Pharmacia, Stockholm, Sweden). The chromatographic system included a C18 silica column and a linear acetonitrile gradient. The advantage of this method is the low detection limit (0.3 microg/ml) combined with detection at 280 nm, which gives a more stable baseline and has less interference from other compounds. As compared to other methods, where shorter wavelengths are used.     

H-point standard addition method for simultaneous spectrophotometric determination of Co(II) and Ni(II) by 1-(2-pyridylazo)2-naphthol in micellar media

A very simple and selective spectrophotometric method for simultaneous determination of Co(II) and Ni(II) by 1-(2-pyridylazo) 2-naphthol (PAN), in micellar media, using H-point standard addition method (HPSAM) is described. The ligand and its metal complexes (Co(II)-PAN and Ni(II)-PAN) were made water-soluble by the neutral surfactant Triton X-100, and therefore, no extraction with organic solvents was required. Formation of both the complexes was complete within 10 min at pH 9 (adjusted by ammonia buffer). The linear range was 0.10-2.00 microg ml(-1) for Co(II) and 0.05-1.50 microg ml(-1) for Ni(II). The relative standard deviation (R.S.D.) for the simultaneous determination of 0.50 microg ml(-1) each of Co(II) and Ni(II) was 2.32 and 3.13%, respectively. Interference effects of common anions and cations were studied and the method was applied to simultaneous determination of Co(II) and Ni(II) in alloy samples. The method was compared with derivative spectrophotometric method.     

Dynamic coating for resolving rhodamine B adsorption to poly(dimethylsiloxane)/glass hybrid chip with laser-induced fluorescence detection

In this paper a method was described about dynamic coating for resolving rhodamine B (RB) adsorption on a hybrid poly(dimethylsiloxane) (PDMS)/glass chip. The results showed that when the non-ionic surfactant Triton X-100 was higher than 0.5% (v/v) into the phosphate buffer, the adsorption of RB appeared. Besides, some separation conditions for RB were investigated, including concentration of Triton X-100, concentration and pH value of running buffer, separation voltage and detection site. Through comparing electroosmotic flow, plate numbers and other parameters, an acceptable separation condition was obtained. Under optimized conditions, the precisions of RB detection (R.S.D., n = 10) were 2.62% for migration time, 4.78% for peak height respectively. Additionally, RB concentration linearity response was excellent with 0.9996 of correlation coefficient between 1 and 100 μM, and a limit of detection (S/N = 3) was 0.2 μM. Finally, we separated rhodamine B isothiocyanate and lysine deriving from the fluorescent probe, and the result displayed that the dynamic coating method was applicable by CE separations using PDMS/glass chip.     

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry

We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-β-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems.     

Determination of 1,4-dioxane impurity levels in Triton X-100 raw material by gas chromatography with mass spectrometric detection

Triton X-100 (octoxynol 9) is a commercially available surfactant used as a solvent detergent in numerous pharmaceutical applications including virus inactivation. A byproduct formed during its synthesis is 1,4-dioxane, the cyclic dimer of ethylene oxide and a possible carcinogen to humans. The United States Pharmacopoeia (USP) contains a labor-intensive 1,4-dioxane test for Triton X-100. The method couples vacuum distillation to extract the 1,4-dioxane from the Triton X-100 matrix followed by gas chromatography (GC) using a packed column with flame-ionization detection. In order to provide a more automated and specific test methodology, a headspace GC-mass spectrometry (MS) method has been developed for this application. Analyte quantitation is accomplished by the method of standard additions. The automated sample preparation, coupled with the specificity inherent in high-efficiency capillary column separations together with single-ion MS detection, results in an assay that is more efficient, accurate, and precise than the USP procedure. Performance characteristics of the headspace GC-MS method are contrasted with those characteristics of the USP methodology.     

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.     

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.     

Self-aggregation and supramolecular structure investigations of Triton X-100 and SDP2S by NOESY and diffusion ordered NMR spectroscopy

The self-aggregation and supramolecular micellar structure of two surfactants in aqueous solution, the anionic surfactant SDP2S (sodium dodecyl dioxyethylene-2 sulfate) and the nonionic surfactant Triton X-100 (octylphenol-polyoxyethylene ether with 9.5 ethoxy groups), were investigated by NMR spectroscopy. The critical micellar concentration (CMC), the size, and shape of the aggregates were determined by diffusion ordered NMR spectroscopy (DOSY), while 2D NOESY NMR spectra were used to study the mutual spatial arrangement of surfactant molecules in the aggregated state. A nonlinear increase of the micellar hydrodynamic radius, indicating possible sphere-to-rod shape transition, was found for SDP2S at higher surfactant concentrations. Triton X-100 micelles were found to be almost spherical at low surfactant concentrations, but formation of ellipsoid shaped particles and/or micellar aggregation was observed at higher concentrations. The NOESY data show that at low concentration Triton X-100 forms a two-layer spherical structure in the micelles, with partially overlapping internal and external layers of Triton X-100 molecules and no distinct hydrophilic-hydrophobic boundary.     

Assessment of polycrystalline graphites as sorbents for solid-phase microextraction of nonionic surfactants

Two polycrystalline graphites (pencil lead and glassy carbon) were used as sorbents for solid-phase microextraction of a nonionic alkylphenol ethoxylate surfactant (Triton X-100). Analyses were performed by reversed-phase HPLC-fluorescence detection. The presence of the benzene ring in the congeners of Triton X-100 also allowed their direct detection at lambda(ex) = 230 nm and lambda(em) = 310 nm. Variables such as time of adsorption, time of desorption and concentration of surfactant in water were evaluated. The method limit of detection was found to be 0.5 microg/l for Triton X-100, with a linear dynamic range of 0.5-150 microg/l. Results were compared to those obtained using polymeric fibers such as PDMS/DVB and Carbowax/TPR. The chemical resistance and low cost of the polycrystalline graphites are advantageous over commercially available SPME fibers.     

Action of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis is significantly influenced by coexisting lipids in substrate-detergent micelles.

The effects of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol on the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail in phosphatidylinositol (PI)/detergent mixed micelles. By addition of PC, the enzymatic hydrolysis of PI was significantly stimulated in PI/Triton X-100 as well as PI/sodium deoxycholate (SDC) mixed micelles. SM stimulated enzyme activity toward PI/Triton X-100 micelles at a lower molar ratio of SM to PI, but was rather inhibitory at a ratio higher than 2.0. The enzyme activity became significantly lower with an increase of PE or cholesterol in PI/Triton X-100 micelles. Actually, both PE and cholesterol were intensively inhibitory when added at a higher molar ratio to PI in Triton X-100-containing micelles. In the system of PI/SDC mixed micelles, not only PC but also SM, PE and cholesterol enhanced the enzymatic hydrolysis of PI. The difference between PI/Triton X-100 and PI/SDC micelles regarding the effects of these lipids on PI-PLC action, must be dependent on the physical state of micelles formed by these detergents and lipids.     

Simultaneous spectrophotometric determination of nitroaniline isomers after cloud point extraction by using least-squares support vector machines

Cloud point extraction has been used for the preconcentration of m-nitroaniline, o-nitroaniline and p-nitroaniline and later simultaneous spectrophotometric determination using polyethylene glycol tert-octylphenyl ether (Triton X-100) as surfactant. The resolution of a ternary mixture of the nitroaniline isomers (after extraction by cloud point) by the application of least-squares support vector machines (LS-SVM) was performed. The chemical parameters affecting the separation phase and detection process were studied and optimized. Under the optimum experimental conditions (i.e. pH 7.0, Triton X-100=0.6%, equilibrium time 20 min and cloud point 75 degrees C), calibration graphs were linear in the range of 0.2-20.0, 0.1-15.0 and 0.1-17.0 microg ml(-1) with detection limits of 0.08, 0.05 and 0.06 microg ml(-1) for m-nitroaniline, o-nitroaniline and p-nitroaniline, respectively. The experimental calibration matrix was designed with 21 mixtures of these chemicals. The concentrations were varied between calibration graphs concentrations of nitroaniline isomers. The root mean square error of prediction (RMSEP) for m-nitroaniline, o-nitroaniline and p-nitroaniline were 0.0146, 0.0308 and 0.0304, respectively. This procedure allows the simultaneous determination of nitroaniline isomers in synthetic and real matrix samples good reliability of the determination was proved.     

Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA–detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.     

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 degrees 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.     

On the localization of water-soluble porphyrins in micellar systems evaluated by static and time-resolved frequency-domain fluorescence techniques

Fluorescence quenching of meso-tetrakis-4-sulfonatophenyl (TPPS(4)) and meso-tetrakis-4-N-methylpyridil (TMPyP) porphyrins is studied in aqueous solution and upon addition of micelles of sodium dodecylsulfate (SDS), cetyltrimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and t-octylphenoxypolyethoxyethanol (Triton X-100). Potassium iodide (KI) was used as quencher. Steady-state Stern-Volmer plots were best fitted by a quadratic equation, including dynamic (K(D)) and static (K(S)) quenching. K(S) was significantly smaller than K(D). Frequency-domain fluorescence lifetimes allowed estimating bimolecular quenching constants, k(q). At 25 degrees C, in aqueous solution, TMPyP shows k(q) values a factor of 2-3 higher than the diffusional limit. TPPS(4) shows collisional quenching with pH dependent k(q) values. For TMPyP quenching results are consistent with reported binding constants: a significant reduction of quenching takes place for SDS, a moderate reduction is observed for HPS and almost no change is seen for Triton X-100. Similar data were obtained at 50 degrees C. For CTAC-TPPS(4) system an enhancement of quenching was observed as compared to pure buffer. This is probably associated to accumulation of iodide at the cationic micellar interface. The attraction between CTAC headgroups and I(-), and repulsion between SDS and I(-), enhances and reduces the fluorescence quenching, respectively, of porphyrins located at the micellar interface. The small quenching of TPPS(4) in Triton X-100 is consistent with strong binding as reported in the literature.     

A fast,sensitive determination of doxorubicin in rat plasma by solid-phase extraction and reversed-phase ion-pair chromatography

A very selective method is described for the determination of doxorubicin in rat plasma. Doxorubicin is extracted from the plasma on a pretreated octadecyl silane column and eluted with phosphate buffer pH 2.6/methanol (25/75, v/v) containing sodium 1-heptanesulfonate as ion-pairing agent. The extraction procedure is suitable for samples which contain doxorubicin encapsulated in liposomes if Triton X-100 is added. A portion of the evaporated eluate is used for high-performance reversed-phase chromatography with the same eluent and a fluorescence detector. Daunorubicin is used as internal standard. Extraction of doxorubicin from plasma is quantitative. The calibration graph is linear for 0.2-100 μg l^?1 doxorubicin with a limit of detection of 0.2 μg l^?1 for 0.5 ml of plasma. The relative standard error of estimate of the calibration was typically 3%.     

Lubrication of the mixed system of Triton X-100/n-C10H21OH/H2O lamellar liquid crystal and ZnS nanoparticles

ZnS nanoparticles could be synthesized, when two kinds of Triton X-100/n-C₁₀H₂₁OH/H₂O lamellar liquid crystal were mixed, in which Zn(CH₃COO)₂ and Na₂S were dissolved in the solvent layer, respectively. The size of ZnS nanoparticles was about 10 nm and limited by the thickness of the solvent layer of the lamellar liquid crystal. The lubrication properties of the mixed system of Triton X-100/n-C₁₀H₂₁OH/H₂O lamellar liquid crystal and ZnS nanoparticles were determined. The results showed that the presence of ZnS nanoparticles could improve the anti-wear ability of the Triton X-100/n-C₁₀H₂₁OH/H₂O lamellar liquid crystal and decrease its friction coefficient.     

Chiral CE separation of dopamine-derived neurotoxins.

An enantiomeric separation of dopamine-derived neurotoxins by capillary electrophoresis has been developed. Tetrahydroisoquinoline (TIQ), dopamine (DA), (R/S)-1-benzyl-TIQ (BTIQ), (R/S)-6,7-dihydroxy-1-methyl-TIQ (salsolinol, Sal), and (R/S)-6,7-dihydroxy-1, 2-dimethyl-TIQ (N-methyl-salsolinol, NMSal) were studied as model compounds. The CE running buffer (50 mM phosphate buffer at pH 3.0) contained 1.5 M urea and 12 mM beta-CD as a chiral selector. During separation, the (R)-enantiomers formed more stable inclusion complexes with beta-CD, and thus had a longer migration time than their optical antipodes. It was noticed that the recovery rates of these TIQ derivatives were very poor (< 15%) during protein precipitation, a procedure widely used for cleaning up biological samples. The recovery was significantly improved by pre-mixing the sample with a surfactant (e.g., sodium hexanesulfonate or Triton X-100) to reduce the co-precipitation. The present method in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied to study samples obtained from in vitro incubation of two catecholamines, dopamine and epinine, with aldehydes forming neurotoxins including (S)- and (R)-NMSal enantiomers. The later is known to induce Parkinsonism in rats.     

Application of renewable silver amalgam annular band electrode to voltammetric determination of vitamins C, B1 and B2

In this work, the design and results of applying silver liquid amalgam film-modified silver solid amalgam annular band electrode (AgLAF-AgSAE), refreshed before each measurement, to voltammetric determination of vitamins C (VC), B₁ (VB1) and B₂ (VB2) are presented. The method is based on adsorptive accumulation of analytes at the AgLAF-AgSAE in a phosphate buffer (VB1), phosphate buffer with Triton X-100 (VB2) and an alkaline borate buffer with Triton X-100 (VC). The analytical parameters and procedure of electrode activation were optimized. The calibration graphs obtained for vitamins C, B₁ and B₂ are linear, respectively, for concentration range 0.05-12, 0.01-0.1 and 0.05-3 mg L⁻¹. The detection limits were calculated and equaled 0.02, 0.003 and 0.009 mg L⁻¹, while repeatability of the peak current was 2%, 1% and 3%, respectively. These results are comparable with results obtained for polarographic determination of the same vitamins using mercury electrodes. Finally, the AgLAF-AgSAE was applied to the determination of vitamins in pharmaceutical samples and fruit juices with satisfactory results.     

Optical properties of Triton X-100-treated purple membranes embedded in gelatin films

The purple membrane (PM) of the microorganism Halobacterium salinarium contains a hexagonally packed monolayer of the light-sensitive protein, bacteriorhodopsin (BR). The optical characteristics of gelatin-immobilized PMs depend strongly on the chemical environment of the PMs in the matrix. Here we present photoinduced absorptive and holographic characteristics of gelatin-embedded PMs solubilized with the non-ionic detergent, Triton X-100. The BR/detergent interaction was shown to slow the M-to-initial state transition of the photocycle and to increase the photosensitivity of the BR films. The lifetime of the holographic grating in Triton X-100-treated BR films was 2–3 times greater, when compared to the unmodified sample. Holographic grating growth times in BR films were shown to change depending on the extent of solubilization. The measured holographic sensitivity appeared to maximize in the range of Triton X-100/BR molar ratios from 15:1 to 25:1. The possible advantages of solubilized PM films as they are applied to optoelectronic devices are discussed.