Dissociation and denaturation behaviour of sesame alpha-globulin in sodium dodecyl sulphate solution.

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, alpha-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of .175-4.0 X 10(-"3) M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 x 10 (-3) M detergent suggesting conformational change; above 8 X 10(-"3) M detergent the value of -[alpha] does not change. The reduced viscosity etared however, increases above .25 X 10(-3) M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an "isoemissive point" at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5-0 X 10(-3) M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and binding constant of 3-0 X 10(3).     

Aggregation, dissociation and denaturation of sesame (Sesamum indicum L.) alpha-globulin in cetyl trimethyl ammonium bromide solution.

The behaviour of the major protein of sesame seed (Sesamum indicum L.) alpha-globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 X 10(-5)--1 X 10(-3) M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 X 10(-2) M CTAB indicate that a conformational change in the protein has taken place.     

Enzymology of ubiquinone-utilizing electron transfer complexes in nonionic detergent.

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied. In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other. Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex-bound micelles in order for enzyme reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary nonenzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex-bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction. With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate-limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate-limiting...     

Influence of nonionic surfactant on aggregation state of scleroglucan in aqueous solution

The interactions between nonyl phenol polyethylene oxide and scleroglucan are investigated by turbidimetry, viscosimetry filterability tests and measurements of elastic modulus and adsorption. The phase diagrams of this ternary system have been established as a function of temperature and composition. It is shown that the surfactant molecules are adsorbed by the polymer at a low surfactant concentration, c_s; the adsorption induces a breaking down of the polymer aggregates and the filterability properties of the solutions are greatly improved. An excess of surfactant phase separation is observed by heating at a temperature that is a decreasing function of c_s. This is explained by the formation of a complex polymer-surfactant which has the same thermodynamic properties (lower critical solution temperature) as polyethylene oxide and the derived nonionic surfactants.     

Mixed micelle formation between gramicidin-S and a nonionic detergent: a nuclear magnetic resonance model study of peptide/detergent aggregation

The interaction of the cyclic decapeptide antibiotic gramicidin-S (GrS) with the nonionic detergent octaethylene glycol mono-n-dodecyl ether was studied by NMR spectroscopy. Detergent binding led to a slightly altered average conformation in the d-Phe side chains of the peptide. The changing diamagnetic shielding of nearby protons resulted in chemical shift variations, the largest effect being observed for the d-Phe C _α proton. The continuous upfield shift of this proton resonance, indicating rapid exchange of the peptide between detergent-associated and unassociated states, was employed for an evaluation of the detergent/peptide aggregation equilibria. The nonlinear binding plot thus obtained was attributed to essentially different aggregational states, depending on the detergent/peptide ratio. The almost linear dependence of the spin-lattice relaxation rate and of the hydrogen-deuterium exchange rate on the fraction of detergent-associated GrS could be reconciled with a simple model, comprising binding of detergent monomers and cooperative binding of micelles at low and high detergent/peptide molar ratios, respectively. Thus, GrS provides a useful model for a study of backbone dynamics and water penetration in detergent- and membrane-bound peptides and proteins. The results will also be discussed with reference to the interaction of GrS with biological membranes. Received: 22 June 1998 / Revised version: 5 October 1998 / Accepted: 9 October 1998     

Removal of nonionic detergent from proteins fractionated by electrofocusing.

The nonionic detergent Nonipol TD 12 (an alkyl polyoxyethylene alcohol) has been removed from solubilised proteins after their fractionation by electrofocusing. Following electrofocusing in nonionic detergent an anionic or cationic detergent was added to the focussing medium and the focusing was allowed to continue. The ionic detergent formed mixed micelles with the nonionic detergent. Thus charged, the mixed micelles migrated to either electrode, removing nonionic detergent from the fractionated proteins. Applying this technique to an adipose tissue preparation, detergent-inhibited activity of a lipolytic enzyme was restored and the binding of adenosine 3':5'-cyclic monophosphate (cyclic AMP) to a protein kinase was increased.     

Lysozyme activity in the presence of nonionic detergent micelles

The effect of a nonionic surfactant, polyoxyethylenesorbitan monolaurate (Tween 20), on the hen egg-white lysozyme catalyzed lysis of a dried cell suspension of Micrococcus lysodeikticus is analysed. A rate enhancement of up to 70% is observed in the presence of surfactant at concentrations above the critical micelle concentration. This activity increase may be explained by postulating the existence of a micelle-enzyme complex in which enzyme molecules are bound to micelles with preferential orientation of their active sites. The reaction is found to be second order with respect to substrate. A mechanism is postulated in which a substrate particle is assumed to be an energy-furnishing collision partner to the enzyme-substrate complex. This mechanism correlated data over a wide range of enzyme and substrate concentrations. Data from kinetic, ultrafiltration, ultraviolet, and fluorescence studies provide convincing evidence for the existence of a micelle-lysozyme complex. The results suggest that it is possible that immobilized enzymes mat in general be more reactive than corresponding free enzymes.     

Value of heptyl-beta-D-thioglucoside, a new nonionic detergent, in studies on membrane proteins

A new nonionic detergent, heptylthioglucoside, was synthesized and found to be more soluble than octylthioglucoside in water at low temperatures. Use of this detergent for solubilization and reconstitution of membrane proteins of Escherichia coli was examined. Heptylthioglucoside was as effective as octylthioglucoside and octylglucoside in solubilizing membrane proteins, and by the heptylthioglucoside-dilution procedure the H+-translocating ATPase (F1F0) and melibiose carrier could easily be reconstituted into liposomes. It is concluded that heptylthioglucoside is very useful in studies on membrane proteins.     

Reactivation of lipid-depleted Ca2+-ATPase by a nonionic detergent.

The Ca2+-ATPase of sarcoplasmic reticulum can be reversibly delipidated by precipitation with polyethyleneglycol in the presence of deoxycholate and glycerol to as low as 4 mol of phospholipid/mol of enzyme polypeptide and can then be reactivated to 90% of its original ATPase activity by the addition of phosphatidylcholine. Furthermore, the preparation exhibits nearly the same activity if the nonionic detergent dodecyl octaoxyethyleneglycol monoether is substituted for the added phospholipid. The delipidated ATPase is soluble in the detergent and retains activity for several days. This is the first report of the Ca2+-ATPase retaining high activity with less than about 30 mol of phospholipid bound per mol of polypeptide.     

Alkaline phytase activity in nonionic detergent extracts of legume seeds

Alkaline phytase activity, with a pH optimum of 8, was recovered from detergent extracts of dormant seeds of nine varieties of Phaseolus vulgaris L., Pisum sativum L. var. Early Alaska, and Medicago sativa L. This alkaline phytase of legume seeds was activated by calcium and differed from most seed phytases in its relative insensitivity to inhibition by fluoride.     

The action of a binary nonionic detergent on a kidney membrane fraction.

The disruption of a kidney cortex microsomal membrane preparation by a binary, nonionic detergent, was followed by using as markers, the changes in total protein content, and (Na+, K+)-ATPase in a supernatant fraction. Both markers responded similarly to changes in pH, microsome concentration and detergent concentration, but responded differently for time-dependent studies. The (Na+, K+)-ATPase activity was increased 2.2-fold (76.1 mumoles Pi/mg protein/h, 95% ouabain-sensitive) by a single detergent treatment and 3.5-fold (92% ouabain-sensitive) by a sequential detergent treatment. Changes in the critical micelle concentration (cmc) were observed for varying detergent and protein concentrations, which suggest interactions of monomeric detergent with the membrane. The peak of (Na+, K+)-ATPase activity occurred above the cmc which suggests the participation of micelles in releasing the enzyme from the membranes. Hill plots of the protein released as the detergent concentration was varied showed a change in the slope near the cmc indicating a four-fold increase in the binding of detergent to membranes as the detergent concentration is increased above the cmc. These results suggest that the disruption of membranes by detergent involves the binding of detergent monomers to the membrane followed by the formation of co-micelles of the detergent with segments of the membrane to complete the separation process.     

Protein extraction and activity in reverse micelles of a nonionic detergent

We describe, for the first time, the ability of a polyoxyethylene sorbitan trioleate-isopropanol microemulsion in hexane to solubilize pure proteins. The dependences of cytochrome c extraction and buffer solubilization by the reverse micellar system on ionic strength of the aqueous phase, detergent concentration, and cosurfactant concentration result in increased extraction. In addition, subtilisin (a serine protease) is shown to be active in this microemulsion. Further the activity of the enzyme can be regulated by the water content of the micelles, enabling control of enzyme activity by "solvent engineering."     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

Increased synthesis of ribonucleic acids in mouse liver after treatment with a nonionic detergent.

A single injection of Tween 40 (polyoxyethylene(20)sorbitan monohexanoate) in the dose range of 600--800 mg/kg body weight induced an increased short-term labeling especially of poly(A)-containing mRNAs in mouse liver (using either [3H]orotic acid or [32P]orthophosphate as RNA precursors), and apparently increased the turnover rates of both rRNAs and mRNAs in this organ over a period of 24 h. In the early period (4 h) after the injection of Tween 40, there was also a significant increase in the content of 32P-labeled adenylic acid in microsomal poly(A)-mRNA fraction. The activity of DNA-dependent RNA polymerases in the nuclei of treated animals was stimulated up to 60% over that in control nuclei in the same period after detergent injection.     

Folding of a monomeric porin,OmpG, in detergent solution

OmpG, a porin from E. coli, has been examined in planar lipid bilayers and in detergent solution. First, bilayer recordings were used to reinforce the evidence that the functional form of OmpG is a monomer. Both pH-dependent gating and blockade by covalent modification add support to this proposal. The findings contrast with the properties of the classical porins, which function as trimers. Second, the folding of OmpG in detergent solution was examined. A water-soluble form of OmpG was obtained by dialysis from denaturant into buffer. Incubation of water-soluble OmpG in detergent results in conversion to a form that possesses the hallmarks of a beta barrel. The folding of water-soluble OmpG in detergent was monitored by circular dichroism, protease resistance, and heat modifiability. OmpG is first transformed into an intermediate with increased beta-sheet content on the time scale of minutes at 23 degrees C. This is followed by the slow acquisition of heat modifiability and protease resistance over several hours. The formation of a beta barrel during this period was demonstrated in a double cysteine mutant by using intramolecular disulfide bond formation to report N and C terminus proximity. Finally, conditions are presented for folding OmpG with greater than 90% efficiency, thereby paving the way for structural studies.     

Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent.

Nonionic detergent (NP40) treatment of paraformaldehyde-fixed normal and SV40-transformed human fibroblasts resulted in intracellular penetration of two chosen fluorescent antibodies and Concanavalin A (Con A). After the detergent treatment nuclear SV40 T antigen, cytoplasmic fibronectin glycoprotein and Con A binding sites could be visualized in fluorescence microscopy. The lowest NP40 concentration which made fixed cells permeable was 0.05%. The morphology of cells was preserved better by this new method than by conventional fixation methods, such as acetone treatment. In scanning electron microscopy the surface of the fixed NP40-treated cells had only small rugosities and fine pores. The subsurface cytoskeleton especially was well preserved and had a more distinct fine structure. The improved morphology made it possible to detect a similar distribution of fibronectin and Con A binding sites in the perinuclear endoplasmic reticulum regions.     

Release of potassium,lipids, and proteins from nonionic detergent treated chicken red blood cells

The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K⁺ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K⁺, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values). © 1994 wiley-Liss, Inc.