DMSO, an Organic Cleanup Solvent for TCA/Acetone-Precipitated Proteins, Improves 2-DE Protein Analysis of Rice Roots

Protein extraction is a critical step in a two-dimensional electrophoresis (2-DE)-based proteomic analysis. Dimethyl sulfoxide (DMSO), a small organic molecule, is widely used as a solvent in biological sciences. In this study, we modified the cleanup step of the commonly used trichloroacetic acid (TCA)/acetone method of protein extraction by using 20?% DMSO/acetone as a solvent to wash TCA?Cacetone-precipitated pellets. The improved protocol (TCA/acetone?CDMSO) was compared with the TCA/acetone and phenol extraction method based on the protein yield, the number of spots, and resolution on 2-DE maps. TCA/acetone?CDMSO washing increased protein yield, and improved the resolution in 2-DE with less background, streaking, and smearing. This method also produced the highest number of protein spots. To our knowledge, the present study is the first to use DMSO as a cleanup solvent for TCA/acetone-precipitated proteins to enhance their quality prior to 2-DE.     

Dimethyl sulfoxide decreases specific EGF binding

Dimethyl sulfoxide (DMSO) stimulates tyrosine phosphorylation of the hepatic EGF receptor in isolated membrane preparations. To determine whether DMSO affects EGF binding, primary cultures of rat hepatocytes were incubated with 1-10% DMSO for 30 min prior to the addition of 125I-EGF. DMSO (1-2%) reduced specific 125I-EGF binding; the effect was maximal (a 40-60% reduction) at 5-7.5% DMSO and was reversed by removing the DMSO. Scatchard analysis showed that the reduction in binding was due to a change in receptor affinity. The decrease in binding was not seen when other, slightly less polar, solvents (eg, acetone and ethanol) were tested. DMSO also reduced 125I-EGF binding to purified rat liver plasma membranes. This reduction was seen in the absence of added ATP and in membranes that had been pretreated with TLCK, a tyrosine kinase inhibitor. Thus, completion of the receptor autophosphorylation reaction was not necessary to effect the change. The data are consistent with a DMSO-induced alteration of receptor conformation that reversibly reduces receptor affinity.     

The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells

Testing of the effects of xenobiotics in cultured cells often requires the use of organic solvents to effect suspension of the test agents in cell culture media. However, the toxic effects of the solvents themselves may introduce artifacts, which obscure interpretation of the experimental results. In this article, the toxicity of different solvents commonly used for solvation of a variety of xenobiotic agents was studied. We show that ethanol, acetone, isooctane, methanol, and hexane were considerably less toxic than the more commonly used solvent, DMSO, when ATP content and growth rates of HeLa cells exposed to these solvents was measured. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 11–15, 1999     

三氯乙酸(TCA)提取法与微波提取法检测活性污泥中三磷酸腺苷(ATP)

【目的】针对活性污泥法中的重要参数ATP进行研究分析,通过在不同条件下检测污泥的活性,得出以ATP为指标的污泥活性状态,为准确判定活性污泥的活性提供依据。【方法】分别运用三氯乙酸(TCA)提取法及微波提取法检测活性污泥中的ATP,并对检测ATP的影响因素(TCA浓度、冰浴时间、p H、微波频率及时间等)进行探讨与优化。【结果】运用TCA提取法检测ATP时,在1.0%-7.0%的TCA体积百分数内,活性污泥中TCA最佳体积百分数为2.5%;在2-60 min的冰浴时间内,最佳冰浴时间为10 min;三羟甲基丙烷-乙二胺四乙酸(Tris-EDTA)缓冲液的最佳p H 7.5;运用微波提取法检测ATP适宜的微波辐射条件为:功率800 W,辐射时间15 s。【结论】TCA提取法和微波提取法均可以检测活性污泥中的ATP,但与微波提取法相比,TCA提取法更能保证从细胞内释放出来的ATP的完整性,因此TCA提取法更适合用于检测活性污泥中的ATP。     

不同实验方法检测常用有机溶剂对细菌活性的影响及其安全使用限量

【目的】采用不同实验方法测定常用有机溶剂二甲基亚砜(DMSO)、丙酮和乙醇对细菌活性的影响,以指导抗菌类药物体外抑菌实验所用溶剂的选择和添加限量。【方法】采用常规体外抑菌实验方法(纸片扩散法、肉汤稀释法),并参照生长曲线法检测有机溶剂DMSO、丙酮和乙醇对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用,采用扫描电子显微镜(SEM)观察溶剂作用后的细菌形态变化。【结果】3种溶剂对E.coli和S.aureus抑菌率达到20%时,在肉汤稀释法下,DMSO、丙酮、乙醇的浓度(体积比)分别为1.00%、0.25%、2.00%和1.00%、1.00%、0.50%;在生长曲线法下,溶剂浓度(体积比)分别为0.50%、1.00%、0.50%和1.00%、0.50%、0.50%;而在纸片扩散法下,32%(体积比)DMSO和32%(体积比)乙醇对E.coli产生明显抑菌圈,但3种溶剂对S.aureus均无抑菌圈出现。3种方法比较后得出:当3种溶剂的抑菌率达到20%时,溶剂浓度(体积比)均低于0.5%,对细菌整体生长活性影响较小。SEM结果表明控制溶剂使用限量可有效减少其对E.coli生长过程的影响。【结论】相对于DMSO和丙酮,乙醇对微生物生长繁殖能力的影响更加明显;采用相同浓度有机溶剂时,液态条件下(肉汤稀释法和生长曲线法)微生物受到有机溶剂的影响较大。     

Evaluation and improvement of spectrophotometric assays of TTC reduction: maize (Zea mays) embryo as an example

The triphenyl tetrazolium chloride (TTC) reduction assay was evaluated and improved with maize seed (Zea mays cv. Zhengdan958). The reduced TTC in embryo was extracted with three kinds of organic solvents: trichloroacetic acid (TCA)/acetone, ethanol, and acetone. The absorbance spectra of the three extracts were similar, with a maximum at 485 nm. The efficiency of TCA/acetone in extracting the reduced TTC was higher than that of acetone and ethanol. A negative correlation between TTC reduction and malondialdehyde content in embryo was demonstrated. The TCA/acetone extraction may be used as a routine protocol for TTC reduction assay of seed vigor in cereal (e.g. maize, rice, wheat and barley) seeds.     

A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP

Cellular ATP is commonly determined as production of bioluminescence using a luciferin-luciferase reaction system. Before the measurement of bioluminescence, cellular ATP must first be extracted. Two commonly used extraction methods are: () Tris-borate buffer (pH 9.2) coupled with a heating process (to inactivate ATPase) and () perchloric acid followed by neutralization. However, we found that both Tris-borate buffer and perchloric acid interfered with the luciferin-luciferase system. Here, we report a convenient single-step boiling deionized water (DW) method for extracting cellular ATP to replace perchloric acid and Tris-borate buffer. We showed that the boiling DW method did not interfere with the bioluminescence and was effective in inhibiting ATPase. This improved method required no neutralization and dilution and thus was more convenient than the perchloric acid method. Unlike the Tris-borate/heating procedure, our method did not require a separate heating step because boiling DW effectively inhibited ATPase and thus accomplished the two missions in one step for both suspended and attached cells. The improved method was precise for both suspended cells and attached cells, when cell numbers were between 10(3) and 10(6). The method also was more sensitive than other methods because it required much fewer cells (10(4) to 10(5)) than other methods for ATP determination. Thus, this one-step method is suitable for routine assay of cellular ATP for both suspended and attached cells.     

Increased transglycosylation activity of Rhodotorula glutinis endo-beta-glucanase in media containing organic solvent

The transglycosylation of p-nitrophenyl-beta-D-cellotrioside to cellotetraose catalyzed by endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50 degrees C in the presence of 40% acetone.     

A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents

Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted disease. To test anti-Trichomonas agents, usually many with low water solubility, organic solvents and surfactant agents should be used. Therefore, the minimal inhibitory concentration (MIC) of acetone, methanol, ethanol, isopropanol, DMSO, Tween 20, Tween 80, and Triton X-100 was determined against T. vaginalis isolates using the quantitative resazurin method. Our results showed that solvents and surfactant agents can be employed as vehicles to test bioactive compounds at lower concentrations than MIC values and we suggest acetone and DMSO as preferential. Moreover, a new methodology is established to substitute or to complement the counting of viable trophozoites. The amount of resazurin reduced by T. vaginalis can be quantified by fluorescence spectroscopy, making the test a quantitative determination of cell viability. These results contribute for pharmacological investigations of bioactive compounds that need the use of solvents as solubilization vehicles to test anti-Trichomonas activity.     

Protein precipitation of diluted samples in SDS‐containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH‐MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample.     

Toxicity of solvents and surfactants to Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) and Dermacentor nitens (Neumann, 1897) (Acari: Ixodidae) larvae

The objective of this work was to verify the sensitivity of Amblyomma cajennense and Dermacentor nitens larvae to the solvents ethanol, methanol, acetone, xylol and dimethyl sulfoxide (DMSO) and the surfactant Tween 80. The first four solvents were tested at analytical purity while the DMSO and surfactant Tween 80 were tested at a concentration of 1%. The substances tested at analytical purity that caused high mortality were also tested at concentrations of 50%, 25% and 1%. The larval packet test was used, with 10 repetitions for each treatment. A control group was also formed with the same number of repetitions, in which the larvae were only exposed to distilled water. In the first experiment, only xylol was highly toxic at the concentrations tested, causing mortality above 90% for larvae of both species. In the second experiment, xylol at 1% and at 25% showed low toxicity to the A. cajennense and D. nitens larvae, respectively, since the percentage mortality was statistically similar to that of the control group (p>0.05).     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.     

Regulation of cellulase synthesis in mycelial fungi: Participation of ATP and cyclic AMP

Summary ATP and cAMP in 4 strains of mycelial fungi were determined by luciferin-luciferase system and HPLC respectively. Cellulase synthesis was subject to the dual control of ATP and cAMP. No matter what carbon sourse was used, cellulase synthesis was repressed if intracellular ATP concentration was over 10^-7mg/ml. Exogenous cAMP could increase cellulase synthesis under depression conditions.     

Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent

A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.     

Determination of susceptibility of Staphylococcus aureus to methicillin by luciferim-luciferase assay of bacterial adenosine triphosphate

M c W alter , P.W. 1984. Determination of susceptibility of Staphylococcus aureus to methicillin by luciferin-luciferase assay of bacterial adenosine triphosphate. Journal of Applied Bacteriology , 56 , 145–150.
Susceptibility of 50 strains of Staphylococcus aureus to methicillin was determined by disc diffusion, minimum inhibitory concentration (MIC) test and luciferin-luciferase assay of bacterial adenosine triphosphate (ATP). ATP concentration time curves calculated on eight strains (four sensitive, four resistant) incubated at 30C indicated 2.5 h as the optimum time for determination of methicillin susceptibility. The ATP concentration and relative light unit (RLU) value of each test broth (antibiotic-containing), expressed as a percentage of its own control broth (antibiotic-free) indicated values of < 30% for ATP and < 40% for RLU to be indicative of methicillin sensitivity. Single time point ATP assays carried out on the 50 strains after 2.5 h at 30C correlated exactly with disc diffusion and MIC. Luciferin-luciferase assay of bacterial ATP appeared to be a reliable, rapid technique for determining the susceptibility of Staph. aureus to methicillin.     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Mutation of PEB4 alters the outer membrane protein profile of Campylobacter jejuni

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.     

Effects of aluminium on ATP pools and utilization in the cyanobacterium Anabaena cylindrica: a model for the in vivo toxicity

ATP pools extracted from the cyanobacterium Anabaena cylindrica , grown in the absence or presence of AlCl₃, were measured using the luciferin-luciferase assay. Addition of low concentrations of AlCl₃ (3.6–36 μ M ) increased the ATP pool 20–40% within 24 h, the effect being more marked with time. When using the Tris-EDTA boiling technique for extraction of cellular ATP, the ATP from aluminium-exposed cells appeared more stable during the extraction than the ATP from untreated cells. The higher ATP pools in aluminium-exposed cells were also evident after dark treatment and addition of the phosphorylating inhibitors carbonylcyanide m -chloro-phenylhydrazone (CCCP) and N,N-dicyclohexylcarbodiimide (DCCD). The formation of elevated ATP pools in cells exposed to aluminium was curtailed by high concentrations of cellular phosphate and postincubation at high pH (>8). These results favour the hypothesis that intracellular aluminium binds to ATP by competing with Mg²⁺ and, as a consequence, the stable Al³⁺-ATP complex formed is no longer available for cellular metabolism. The cyanobacterium is assumed to compensate by increasing the total pool of ATP. At high AlCl₃-concentrations, and in particular at low phosphate: aluminium molar ratios (<1), aluminium apparently also interferes with the membranes in A. cylindrica as indicated by inhibited O₂ production, reduced ATP production and cell lysis.     

Kinetics of bactericidal activity of antibiotics measured by luciferin-luciferase assay

ATP production, measured by the luciferin-luciferase assay, is an indicator of bacterial metabolic activity. This enzymatic assay yields rapid results (< 5 minutes), permitting multiple measurements and establishment of ATP growth curves in order to study the kinetics of antibiotics in bacterial populations. The measurement of free or extracellular ATP, total ATP (extra and intracellular) and the ratio of free to total ATP are additional means of studying the bacteriostatic or bactericidal activity of antibiotics. An increase in free ATP is an indicator of extracellular movement due to alteration of the cell wall. The ratio free ATP/total ATP × 100 ≥ 50%, indicates bacteriallysis. These assays were used to study the effects of 14 antibiotics on two reference strains of Escherichia coli ATCC 25922 and Staphylococcus aureus 25923.