The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% (v/v) aqueous solutions of the detergent contained 0.4 to 3.2 microM equivalent peroxide but iodometric titration revealed 1.1 to 5.0 microM peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 X 10(7) M-1 S-1 at pH 7.4 at 25 degrees C, and for conversion into Compound II apparent first-order rate constants were 5.2 X 10(-3) to 1.7 X 10(-2) S-1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.     

Resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products.

The inactivation of enveloped viruses by two different solvent/detergent combinations, i.e. tri-n-butyl phosphate (TNBP)/Triton X-100 or TNBP/Tween 80, has been investigated using a high purity factor VIII (Replenate) and factor IX (Replenine) respectively. Treatment with TNBP/Triton X-100 rapidly inactivated all the typical enveloped viruses tested, i.e. Sindbis, semliki forest virus (SFV), herpes simplex virus type-1 (HSV-1) and vesicular stomatitis virus (VSV), by 3.7-5.8 log within 15 seconds. While virus inactivation with TNBP/Tween 80 was slower, effective inactivation of Sindbis, HSV-1, VSV and human immunodeficiency virus type-1, i.e. 4.1-->6.3 log, occurred within 30 minutes. In contrast, vaccinia virus was relatively resistant to inactivation in either of these solvent/detergent combinations. Incubation times of 10 minutes for TNBP/Triton X-100 or 6-24 hours for TNBP/Tween 80, were required to reach inactivation levels of about 4 log.     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.     

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

表面活性剂对林可霉素发酵生产的影响

在林可霉素发酵过程中,当向培养基中加入表面活性剂十二烷基磺酸钠(SDS)、吐温80(Tween 80)和曲拉通(Triton X-100)时,林可霉素的产量受到较大影响。本研究应用响应面设计法(Response surface design)对表面活性剂的配比进行了优化,得到的优化配比为:十二烷基磺酸钠为31.13 mg/100 mL,吐温80为51.97 mg/100 mL,曲拉通为16.9 mg/100 mL。将该优化配比应用于林可霉素发酵,产量提高了36.67%。     

Comparison of particulate 3,3',5,5'-tetramethylbenzidine and 3,3'-diaminobenzidine as chromogenic substrates for immunoblot

In horseradish peroxidase (EC: 1.11.1.7)-dependent immunoblot assays, particulate 3,3',5,5'-tetramethylbenzidine (TMB) is shown to be a more efficient immunoblot substrate than the standard substrate 3,3'-diaminobenzidine (DAB), because TMB is easily prepared, stable, and less carcinogenic than is DAB. Assays of antibody in a serially diluted human immunodeficiency virus (HIV) control serum (CDC reference CAT# VS2151) have the same sensitivity limits with both DAB and TMB (1:312,500). Complete, working substrate solutions of H2O2/TMB/enhancer and of H2O2/DAB were stored at room temperatures and at 48 degrees C respectively. Periodic tests showed the TMB substrate system to be functional after four weeks at 48 degrees C and after eight weeks at room temperature, while the DAB system was functional after one week at 48 degrees C and after four weeks at room temperature. The stability, safety, and convenience of the commercially available TMB kits make this substrate ideal for immunoblot tests.     

Toxicity of phenanthrene dissolved in nonionic surfactant solutions to Pseudomonas putida P2

The fraction in which direct contact occurs between micellar-phase phenanthrene and the bacterial cell surface was estimated by measuring the toxicity of nonionic surfactant (Tween 80 and Triton X-100) solutions to the phenanthrene-degrading bacterium, Pseudomonas putida P2. Cell viability of completely dissolved phenanthrene decreased by 30% at concentrations greater than 0.3 mg L(-1), which is equal to approximately one third of its solubility. Both nonionic surfactants had no effect on cell viability up to 5 g L(-1). Cell viability increased with increasing surfactant concentration at a fixed phenanthrene concentration, due to the decreased concentration of aqueous-pseudophase phenanthrene and the reduced fraction of direct contact. The fraction of direct contact was c. 20% or more below 3 g L(-1) of Triton X-100. The fraction of direct contact for Tween 80 was estimated to be lower than Triton X-100.     

Effect of surfactants on ethanol fermentation using glucose and cellulosic hydrolyzates

The effect of the non-ionic surfactants on the ethanol fermentation was greatly dependent on the surfactant added. While Tween 20 and Tween 80 slightly enhanced ethanol fermentation, Triton X-100 which exhibited the inghest increase in the enzymatic saccharification had a negative effect on the ethanol fermentation. The negative effect of Triton X-100 on ethanol production was the most pronounced when the cellulosic hydrolyzates were used. Tween 80 showed the best performance for the ethanol production from steam exploded wood hydrolyzate.     

Effect of surfactants on cellulase production by Nectria catalinensis

The effect of different nonionic surfactants (Tween 80, Tween 20, Triton X-100) and polyethylene glycol (PEG 6000) was tested on cellulolytic enzyme system production. Tween 80 gave the highest yield of endoglucanase, exoglucanase, and cellobiase at the 20th day of growth, presumably by causing increased permeability of cell membranes and/or by promoting the release of cell-bound enzymes. Maximal yield of endoglucanase was achieved with 1.7 mM Tween 80, whereas exoglucanase and cellobiase were at 0.85 mM. In the same way, this compound increased fungal growth. On the other hand, Tween 20 and Triton X-100 inhibited growth and cellulolytic enzyme production. High yields of endoglucanase and exoglucanase were achieved with PEG 6000 in comparison with the control, presumably by increasing enzyme stability. Received: 22 January 1996 / Accepted: 28 March 1996     

Effects of Four Detergents on the Oxidative and Phosphorylating Capacities of Potato Mitochondria

Oxidative and phosphorylating capacities of potato mitochondriawere studied with an oxygen electrode; succinate was used asa respiratory substrate. When low concentrations (0·1mg ml^–1 or less) of four detergents (Triton X-100, Tween80, sodium cholate, and sodium deoxycholate) were added intothe medium, the respiratory rates, respiratory controls, andADP/O ratios of mitochondria were increased. At higher concentrations,Triton X-100 and sodium deoxycholate were sharp inhibitors ofthe mitochondrial respiration; sodium cholate and over all Tween80 were much less toxic for potato mitochondria.     

Quantitation of human immunoglobulin G and albumin in electroimmunodiffusion gels containing ionic and nonionic detergents

Quantitation of human immunoglobulin G (IgG) and albumin by agarose electroimmunodiffusion is influenced by the incorporation of ionic and nonionic detergents in the gel. The highest concentrations of each detergent at which human IgG and albumin determinations could be performed without perturbing the quantitations were 4% Triton X-100, 4% Tween 80, 1% NP-40, 0.5% sodium deoxycholate (SDOC), 0.5% Zwittergent, and 0.1% sodium dodecyl sulfate (SDS), and mixtures of Triton X-100, SDOC, and SDS. These detergent combinations all resulted in greater perturbations of albumin quantitation than of IgG. Immunoprecipitation of human IgG was quantitated in the absence and presence of Triton X-100, Zwittergent, and SDS. SDS was shown to cause nonspecific precipitation, whereas below 1% Triton X-100 or 0.5% Zwittergent no effects upon the immunoprecipitations were observed.     

Optimization of mono and diacylglycerols production from enzymatic glycerolysis in solvent-free systems

This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 °C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.     

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

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

Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle

The dihydropyridine receptor-calcium channel complex, prelabeled with (+)-[3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86-87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein.     

Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labelled proteins

In order to establish optimum conditions for the chemiluminescent (CL) reaction of two acridinium ester labelled proteins (human albumin and rabbit anti-human albumin IgG), we investigated the effects of the following factors known to influence the CL emission: pH, presence of proteins, relative concentrations of components of CL reaction and presence of surfactants. Under optimal conditions of pH and hydrogen peroxide concentration, hexadecyl trimethyl ammonium chloride (CTAC) increased the intensity of the CL reaction of the acridinium ester labelled albumin by 42-fold. Triton X-100, Tween-20, 23 lauryl ether (Brij 35) and sodium dodecyl sulphate (SDS) exerted a much smaller effect. In the case of the acridinium ester labelled antibody, the greatest increase was obtained with Triton X-100 (15-fold) followed by CTAC, Brij 35 and Tween 20 (SDS decreased the emission intensity).     

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

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

Influence of surfactants on growth and regeneration from mature internodal stem segments of sweet orange (<Emphasis Type="Italic">Citrus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis>) cv. Hamlin

The development of a reliable shoot regeneration system for mature tissue of citrus is of major importance to accelerate the evaluation of commercial traits. Three non-ionic surfactants were evaluated independently in terms of their affects on the growth and regeneration of mature internodal stem segments of sweet orange cv. Hamlin in culture. Growth and shoot development of explants were influenced by type of surfactant added to the regeneration medium DBA3, its concentration and order of flush growth used for explant preparation. Supplementation of Pluronic F-68 at 0.001% (w/v) to the medium was the superior treatment resulting in significantly higher fresh weight gain of explant, improved mean number of shoots per explant and the percentage of explants giving shoots (33.5% from first flush) and shoot yield was twofold higher compared to treatments without surfactant (17%). Triton X-100 was the least responsive in terms of its affect on the growth and regeneration of stem segments but such shoots had a normal phenotype. Explants cultured on DBA3 medium containing Tween 20 exhibited growth and shoot yield similar to treatments without surfactant, but at concentrations 0.01–0.5% (v/v), the shoots became vitrified and failed to graft successfully in vivo. Growth and shoot yield of explants showed a general decline between flushes especially from second and third harvests. Shoots derived from stem segments which were cultured on media containing Pluronic F-68 and no surfactant had a higher survival rate (70–80%, respectively) compared to treatments using Triton X-100 at 0.001–0.1% (v/v) (33% survival). All acclimatized grafts exhibited typical mature wood characteristics and flowered 14–16 months after transfer to the greenhouse.     

基于全细胞催化合成NADP重组细胞复合通透剂的筛选

本文以重组大肠杆菌E. coli BL21(DE3) p ET30α(+)-Nmnat为研究对象,考察化学通透剂处理重组菌对其催化合成NADP的影响。实验显示,在单因素筛选实验中,四种通透剂曲拉通X-100、吐温80、二甲基亚砜和十六烷基三甲基溴化铵(CTAB)对细胞进行通透化处理均可提高全细胞催化产率,且提高幅度差距并不显著。进一步采用析因设计考察四种通透剂的复合效应,除了曲拉通X-100与吐温80和曲拉通X-100与CTAB交互作用的P大于0. 05为不显著,其余所有交互作用项P值均小于0. 05,呈现显著性,且复合通透剂中,仅单一增加CTAB量,复合体系的协同作用将减少,制约作用增加,不适宜用于复合配方。最后采用中心组合设计进一步对复配效果进行优化,通过拟合得到最佳通透剂配比为曲拉通X-100(1. 64%),吐温80(2. 53%)以及二甲基亚砜(1. 06%),此时NADP预测产率达到77. 46%,与实际验证产率均值77. 05%相差甚微,说明实验优化模型具有良好预测性,优化结果与未添加通透剂相比提升近47%,表明优化复合通透剂配方更有利于提高重组菌催化合成NADP。     

The effect of some biphenyl solubilizing and suspending agents on biphenyl 4-hydroxylase of rabbit liver microsomes

The effects of ethanol, acetone, dimethylsulfoxide (DMSO), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), Triton X-100, and carboxymethyl cellulose (CMC) on the kinetics of biphenyl 4-hydroxylase of rabbit liver microsomes were investigated in an attempt to find a substrate-solubilizing or suspending agent (carrier) which was itself a non-effector of the mixed-function oxidase. The effects of these carriers on the activities of NADPH-cytochrome P-450 reductase, NADPH-cytochrome c reductase, and cytochrome P-450 content were also investigated.Ethanol and DMSO inhibited biphenyl 4-hydroxylase and NADPH-cytochrome P-450 reductase. Acetone inhibited the hydroxylase uncompetitively at concentrations which appeared to stimulate NADPH-cytochrome P-450 reductase. All of the detergents inhibited biphenyl 4-hydroxylase although only Triton X-100 markedly affected the reduction of cytochrome P-450. The interaction of Tween 80 with the hydroxylase gave rise to non-linear Lineweaver-Burk plots although at high concentrations of biphenyl or low concentrations of the detergent the inhibition appeared to be competitive.Biphenyl caused a 2–3-fold stimulation of NADPH-cytochrome P-450 reductase, but in the presence of Tween 80 the stimulation was absent. Since V of biphenyl 4-hydroxylase in the presence of Tween 80 was not significantly different from V in its absence it would appear that the reduction of cytochrome P-450 was not ratelimiting.Of all the carriers studied only CMC was without effect on all aspects of microsomal electron transport investigated. As far as biphenyl 4-hydroxylase is concerned, CMC appears to be the most suitable substrate carrier.