A fluorescamine assay for submicrogram quantities of protein in the presence of Triton X-100

The sensitivity of the fluorescamine assay is increased by hydrolysis of the protein sample in 2 n NaOH. The assay is linear from 0.125 to 10 μg of protein and can measure concentrations as low as 0.5 μg/ml. The presence of several detergents including Triton X-100 in both the protein sample (up to 10%) and in the fluorescamine assay itself (up to 0.33%) does not interfere.     

Estimation of labile sulfide in iron-sulfur proteins.

Lowry's method (1) for protein determination is subject to interference from the nonionic detergent Triton X-100 (2,3) which is used in high concentrations (1–5%) to solubilize membrane proteins or enzymes (4–6) and structural acidic proteins (7). Hartree (3) could reduce the errors caused by 0.1% Triton X-100 by a modification of Lowry's method. However, when protein solutions containing 0.2% or more of the detergent are mixed with the Folin-Ciocalteu reagent (1) a precipitate forms that interferes with the assay. We could reduce this interference to an insignificant level either by centrifuging the precipitate and incorporating Triton X-100 in both the reagent blank and standards, or by removing the detergent prior to the assay. This report presents two simple procedures for the Lowry assay of dilute protein samples containing 1–5% Triton X-100.     

Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis.

A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Rapid protein release from Escherichia coli by chemical permeabilization under fermentation conditions

Overall protein release greater than 75% in less than 1 h can be attained by exposing exponentially growing Escherichia coli cells to 0.4 M guanidine plus 0.5% Triton X-100 at 37 degrees C in medium. Cell growth stops immediately upon addition of the chemicals, but the cells are not lysed. Guanidine concentrations lower than 0.2 M, in conjunction with 0.5% Triton X-100, do not release significant intracellular protein, nor do they inhibit cell growth. Under these conditions, the cells undergo an adaptation that confers resistance to protein release by further treatment with guanidine and Triton X-100. Cells treated with 0.2 M guanidine plus 0.5% Triton X-100 display intermediate behavior. Protein release is approximately 35%, and growth is temporarily interrupted by an extended lag phase. Subsequent resumption of cell growth results in resistant cells and no additional protein release. This resistance is shown to be reversible and is most likely due to physiological adaptation rather than genetic mutation.     

Dependence of yeast permeabilization with Triton X-100 on the cell age

Summary Acid phosphatase activity and protein release were determined in cell suspensions ofYarrowia lipolytica andTorulospora delbrueckii at different stages of their growth by permeabilization with Triton X-100. The effect of the surfactant on the cell permeability did not depend on the cell age.     

In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.     

A procedure for the estimation of microgram quantities of triton X-100

A spectrophotometric procedure is reported for the assay of microgram amounts of the nonionic detergent Triton X-100. The method is based on the reaction of ammonium cobaltothiocyanate with the poly (ethylene oxide) groups of Triton X-100 to form a blue precipitate. The latter is extracted into ethylene dichloride and assayed spectrophotometrically. Capable of assaying as little as 40 μg of Triton X-100, the procedure is applicable in the presence of proteins provided a small easily determined correction is applied. High ionic strength (up to 2 m NaCl tested) did not interfere with the method.     

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.     

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.     

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.     

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.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the cytoskeleton fractions of rat red blood cells prepared with non-ionic detergents

The characteristics of cytoskeleton fractions prepared from rat red cell ghosts with four non-ionic detergents were studied. One percent (w/v) solutions of Triton X-100, Emulgen 911, MEGA-9 (nonanoyl-N-methylglucamide), and octylglucoside solubilized 78, 68, 80, and 92% of the ghost phospholipid, while they solubilized 82, 78, 72, and 62% of the ghost band 3, a transmembrane protein, respectively. There was no correlation between the solubilization percentages of phospholipid and band 3. Phospholipids retained in cytoskeleton fractions were shown to exist as blebs on the surface by electron microscopic observation. The cytoskeleton fraction prepared with octylglucoside retained about two-fold more band 3 than that with Triton X-100 (Triton shells). However, cytoskeleton fractions prepared from p-chloromercuribenzoate-treated ghosts with the two detergents retained almost equal amounts of band 3, less than 5% of that in the ghosts. Under this condition, most of band 2.1, a protein linking band 3 to the spectrin-actin network, was released from the cytoskeleton fractions. The band 3 solubilized with octylglucoside sedimented faster in a linear sucrose gradient and had a larger Stokes' radius than that with Triton X-100, which is known to exist as dimer. These results strongly suggest that octylglucoside does not disturb the association of tetrameric band 3 with the spectrin-actin network, while Triton X-100 dissociates tetrameric band 3 to the dimer, resulting in the difference in the amount of band 3 retained in cytoskeleton fractions. In conclusion, octylglucoside can produce a more native cytoskeleton fraction of red cell membranes than Triton shells.     

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.     

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.     

Evidence for a membrane-bound fraction of chick intestinal calcium-binding protein

After homogenization of intestinal mucosa from vitamin D-replete chicks and high speed centrifugation, the major proportion of the vitamin D-induced calcium-binding protein is present in the supernatant fraction. However, the centrifugate, after repeated washing, contains significant amounts of bound calcium-binding protein that can be solubilized by Triton X-100. The bound calcium-binding protein is identical to soluble calcium-binding protein by the criteria of immunological identity, electrophoretic mobility, and molecular size, as determined by gel filtration chromatography. The bound calcium-binding protein is only partially released by sonication, osmotic shock or by ribonuclease treatment Bound and soluble calcium-binding protein are not present in rachitic chick intestine. The addition of calcium-binding protein to rachitic mucosa prior to homogenization does not yield a Triton X-100 solubilizable form, indicating that bound calcium-binding protein in vitamin D-replete intestine is not due to adsorption of vesicular entrapmetn of soluble calcium-binding protein. The overall evidence suggests that part of the intestinal calcium-binding protein is membrane-bound.     

The size and detergent binding of membrane proteins.

Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.     

T cell receptor can be recruited to a subset of plasma membrane rafts,independently of cell signaling and attendantly to raft clustering

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3epsilon, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3epsilon amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca(2+) mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM(1). We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.     

The solubilization and morphological change of human platelets in various detergents

The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen.