Deleterious effect of Brij 35 on alkyl 2-pyrones and other hydrophobic inhibitors of human sputum and leucocyte elastase

Brij 35 significantly reduced the inhibitory activity of hydrophobic alkyl 2-pyrones, oleic acid and alkyl peptides towards human sputum and leucocyte elastase, whereas 4-methoxy-6-(2'-hydroxy-2'-(carbobutyloxy)-vinyl)-2-pyrone, alpha-1-proteinase inhibitor and a sulfated chitosan were unaffected. The effect of Brij 35 on elastase appeared to be irreversible, since dialysis against Brij-free buffer was not accompanied by a return to inhibitory activity by the first group of inhibitors. However, passage through an ionic-exchange column was effective in removing the detergent from the enzyme. Brij 35 is also an activator of the elastases: kcat for Boc-Ala-4-nitrophenyl ester and methylsuccinyl-Ala-Ala-Pro-Val-4-nitroanilide increased by 20% and 40%, respectively in the presence of 0.015% Brij 35. Binding of the substrates to the enzyme is unaffected, since Km is unchanged.     

The multidrug resistance protein ABCC1 drug-binding domains show selective sensitivity to mild detergents

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [125I]iodoaryl azido-rhodamine123 (IAARh123)-a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-beta-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1-drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

A simple peptide mapping method by partial filling micellar electrokinetic capillary chromatography with a zwitterionic-nonionic mixed micelle

A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) method with a mixed micelle system composed of a zwitterionic surfactant named 3-(N,N-dimethylhexadecylammonium)propanesulfonate (PAPS) and a nonionic surfactant polyethylene glycol dodecyl ether (Brij 35) for peptide mapping is described. The method was demonstrated by the separation of tryptic digestion of bovine serum albumin (BSA). The optimal mixed micelle solution was 50 mM NH(4)OH-HCOOH buffer (pH 2.0) containing 32 mM PAPS and 0.6% (m/v) Brij 35. It was found that the mixed micelle system permitted a highly selective separation of the tryptic digestion. The high separation selectivity was probably due to the ion-pairing interaction between the zwitterionic surfactant molecules and the peptides.     

Crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum

Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.     

Multiple forms of biliverdin reductase: modification of the pattern of expression in rat liver by bromobenzene

Rat liver biliverdin reductase was purified from control and bromobenzene-treated rats and was designated as C-BVR-T and B-BVR-T, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the existence of two molecular weight variants (30,100 and 29,800) in C-BVR-T but only one form (30,100) in B-BVR-T. Western immunoblotting confirmed that both molecular weight variants were biliverdin reductase. Nondenaturing electrophoresis separated C-BVR-T and B-BVR-T preparations into groups of four variants, designated as BVR ND1 to ND4. However, the C-BVR-T preparation contained three major forms (BVR ND1, ND2, and ND3) while the B-BVR-T preparation contained two major forms (BVR ND2 and ND3). In vitro treatment of biliverdin reductase preparations with either bromobenzene or dithiothreitol did not interconvert the variants of the enzyme. QAE-Sepharose anion-exchange chromatography was used to isolate the ND2 and ND3 variants for physiochemical analysis. The amino acid composition of the variants was rather similar except for their Tyr content. Also, the peptide maps were similar except for a series of moderately early chromatographic peaks. These findings implied secondary modifications to the protein rather than substantial differences in primary structure. The pH-dependent cofactor requirements for enzyme activity were examined. Both variants exhibited 2 pH optima that were cofactor dependent; maximum activity with NADPH and NADH was observed at pH 8.5 and 6.7, respectively. However, both variants exhibited a higher catalytic rate with NADH than with NADPH at their pH optima. Furthermore, BVR ND3 exhibited a higher catalytic rate than BVR ND2 with either cofactor throughout the pH range 6.5-9.     

Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80–90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H⁺-ATPase and the Ca²⁺-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C₁₆E₂₀) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80–90% right-side-out vesicles. This was shown by assaying ATP-dependent H⁺ pumping using the ΔpH probe acridine orange and dissipating the H⁺ gradient with nigericin, and by assaying ATP-dependent Ca²⁺ transport using ⁴⁵CA²⁺ and dissipating the Ca²⁺ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E_20–23) showed this 'non-detergent behavior', whereas those with smaller head groups (E_8–10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.     

A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gels

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H₂O₂ equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe²⁺ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO₃ or SnCl₂ followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.     

Use of cyclodextrins as chiral selectors for direct resolution of the enantiomers of fluoxetine and its metabolite norfluoxetine by HPLC

The goal of this work is to investigate the direct chromatographic separation of the enantiomers of fluoxetine and its active metabolite norfluoxetine. The liquid chromatographic retention behavior of these enantiomers on a β-cyclodextrin bonded-phase column was investigated with respect to mobile phase composition, pH, ionic strength, and solvent selectivity. Relationships were established between these factors and the three most important chromatographic parameters: retention time, resolution, and selectivity. Most of the evidence suggests that the unique selectivity of this column isdue to inclusion complex formation, which provides the physical basis for enantiomeric resolution. After these studies a set of optimum chromatographic conditions was chosen for the simultaneous separation/determination of a mixture of the four enantiomers using fluorescence detector. © 1993 Wiley-Liss, Inc.     

Study of the Effect of Surfactants on Extraction and Determination of Polyphenolic Compounds and Antioxidant Capacity of Fruits Extracts

Micelle/water mixed solutions of different surface active agents were studied for their effectiveness in the extraction of polyphenolic compounds from various varieties of apples from west Azerbaijan province in Iran. The total content of polyphenolic compound in fruit extracts were determined using ferrous tartrate and Folin–Ciocalteu assays methods and chromatographic methods and compared with theme. High performance liquid chromatography is one of the most common and important methods in biochemical compound identification. The effect of pH, ionic strength, surfactant type, surfactant concentration, extraction time and common organic solvent in the apple polyphenolics extractions was studied using HPLC-DAD. Mixtures of surfactants, water and methanol at various ratios were examined and micellar-water solutions of Brij surfactant showed the highest polyphenol extraction efficiency. Optimum conditions for the extraction of polyphenolic compounds from apple occurred at 7 mM Brij35, pH 3. Effect of ionic strength on extraction was determined and 2% (W/V) potassium Chloride was determined to be the optimum salt concentration. The procedure worked well with an ultrasound bath. Total antioxidant capacity also was determined in this study. The method can be safely scaled up for pharmaceutical applications.     

Simultaneous determination of three local anesthetic drugs from the pipecoloxylidide group in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C(18) reversed-phase column (150 x 4.6 mm I.D.) filled with 5-microm particles and attached to a UV detector. The mobile phase was composed of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88-91% for mepivacaine, 87-89% for ropivacaine and 88-91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.     

Assay for the quinocarmycin analog DX-52-1 in human plasma using high-performance liquid chromatography with automated column switching and low wavelength ultraviolet detection

The hydrocyanated derivative of the antitumor antibiotic quinocarmycin, DX-52-1 (I), exhibits impressive activity against human melanoma xenograft models in vivo. Phase I clinical trials to evaluate this compound as an antineoplastic agent have been initiated by the US National Cancer Institute. We have developed an HPLC assay for the determination of I in human plasma involving automated column switching and UV detection at 210 nm. The preparation of samples for chromatographic analysis entails the preliminary removal of plasma proteins by precipitation with acetonitrile, acidifying the clear supernatant to pH 4.5, then extracting twice with tert.-butyl methyl ether to recover the drug. A heart-cutting procedure employing two HPLC columns with contrasting retention characteristics under isocratic reversed-phase conditions was used to achieve the selectivity required for low wavelength UV detection of the analyte. The sample extract was initially loaded onto a column packed with a cyanopropyl stationary phase. During the predetermined time interval that I eluted from this column, a fully automated six-position switching valve was used to direct the effluent onto an octadecylsilane analytical column. The assay has been thoroughly validated with regard to linearity, inter- and intra-day accuracy and precision, recovery, selectivity and specificity. Using a sample volume of 1.0 ml, the lowest concentration of I quantified with acceptable day-to-day reproducibility was found to be 2.56 ng/ml (R.S.D. 18.9%, n=21, 4 months). This proved to be sufficiently sensitive for pharmacokinetic drug level monitoring in cancer patients treated with a 6-h continuous intravenous infusion of I, even at the starting dose of 3 mg/m². The successful performance and reliability of the assay has been demonstrated through extensive application to the routine analysis of plasma specimens acquired during a phase I clinical trial of the drug.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

Preparation and properties of soluble, immunoreactive apoLDL.

Immunoreactive apo-low density lipoprotein (LDL), soluble in mildly alkaline buffers of low ionic strength, was prepared by attaching LDL to a DEAE-Sepharose column and eluting the lipids with a 0--2% (w/v) gradient of nonionic detergents. Brij-36T, Nonidet P-40, and Triton X-100 gave similar results. After washing the detergent from the column, the bound apoLDL was eluted with 1 M NaCl, pH 7.4, with recoveries up to 85%. This apoLDL could be dialyzed extensively against low ionic strength solutions, and remained soluble as long as the pH was above 7. Spectrophotometric analysis showed that less than 0.1% %w/v) of cholesterol or phospholipids and less than 1% (w/v) of detergent remained associated with the protein. The apoLDL cross-reacted with LDL against antisera prepared vs. intact LDL. Pore-gradient polyacrylamide gel electrophoresis, with SDS and urea, showed that this preparation was less aggregated than organic solvent extracted apolLDL and appeared to be made of oligomers of two monomeric subunits, one with molecular weight around 22,700 and a smaller one of approximately 8000. Isoelectric focusing showed that there also was charge heterogeneity in the soluble apoLDL.     

Interactions of melittin, a preprotein model, with detergents.

Bee venom melittin is a water-soluble tetramer of identical polypeptide chains. Each chain has 26 residues. The 20 N-terminal residues are hydrophobic and the 6 C-terminal residues are basic. Melittin has been shown to integrate into natural and synthetic membranes and to lyse a wide variety of cells. To understand how a water-soluble protein can spontaneously partition into a membrane, we have studied the interaction of melittin with micelles of deoxycholate (DOC), Brij 58, and sodium dodecyl sulfate (NaDodSO4). Circular dichroism spectra showed that NaDodSO4, an ionic detergent, and Brij 58, a nonionic detergent, caused similar major changes in the protein's conformation. Gel filtration studies revealed that melittin forms mixed micelles with either Brij or DOC. The melittin-DOC mixed micelles have 2 mol of DOC per mol of melittin. Cross-linking studies with dimethyl suberimidate confirmed that the protein is a tetramer and showed that it becomes monomeric either in mixed micelles with Brij or DOC or in butanol. Despite this major structural change of melittin in the presence of an amphiphile, the covalently cross-linked form is as active in human erythrocyte lysis as the native protein.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

Stabilization and crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum

Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.     

Protection of sodium dodecyl sulfate-induced aggregation of concanavalin a by saccharide ligands

Concanavalin Å is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin Å requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl β-d-galactopyranoside did not inhibit the aggregation of concanavalin Å by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin Å may have an additional, ligand-binding site which is netal-dependent and which can be modified by the addition of a saccharide ligand.     

A structural characterization of gap junctions isolated from mouse liver

Mouse liver gap junctions have been isolated by using an anionic detergent, n-dodecanoyl sarcosine, in combination with non-ionic polyoxyethylene ethers (Brij 35 and Brij 58) and (W-1) detergents. Purified gap junctions are obtained in a sucrose step gradient containing 1-o-n-octyl-beta-D-glucopyranoside. This procedure is aimed at minimizing proteolysis. The junctions thus isolated have a hexagonal lattice of connexons with a lattice constant of 7.6-8.4 nm. Presence of a major Mr 26,000 gap junctional protein has been confirmed by SDS-PAGE.