A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for determination of methionine containing radioactivity in the thiomethyl moiety

A method is described for the determination of methionine containing a radioactive label in the thiomethyl moiety. Cyanogen bromide in aqueous acetic acid is used to convert the labeled thiomethyl moiety to labeled methylthiocyanate which is then extracted into toluene and counted by liquid scintillation.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

A note on two liquid scintillation fluors useful for primary production work

Summary Two liquid scintillation fluors, the first using a 2-methoxyethanol/toluene solvent, the second a Triton X-100/toluene solvent, are discussed. Data presented indicate that the technique using the 2-methoxyethanol/toluene solvent produces higher d.p.m. than does solid support counting. The Triton X-100/toluene solvent fluor is suggested for measuring aqueous samples such as algal excretion products and ¹⁴C stock solutions.     

A simple method for separating unbound and bound cortisol in a radioimmunoassay

A cortisol radioimmunoassay in which unbound cortisol is partitioned into the organic phase of a toluene: water scintillation fluid mix at 0 to 5 degrees C is described. Antibody-bound cortisol remained in the aqueous phase. Since liquid scintillation spectrometers detect photons generated from the [3H]cortisol only in the organic phase, the system effectively separates antibody bound from unbound [3H]cortisol. Regression coefficients including linear, quadratic, and cubic components of standard curves were between 0.980 and 0.999. Cross-reactivity was 3% or less with 11 other steroids and cholesterol except for cortisone (16%) and prednisone (12%). Intra- and interassay coefficients of variation were 8 and 13%, respectively. The lower limit of sensitivity of the assay was 1.4 ng/ml. Recoveries of added mass averaged 97.5%. The correlation between concentrations of glucocorticoids assayed by competitive binding to dog plasma and the current procedure was 0.90. The assay procedure described simplifies separation of unbound from antibody-bound cortisol.     

A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.     

A new partition thin-layer chromatographic method for steroid separations

TLC plates with a 25 mu thick polyamide stationary phase were modified for the separation of neutral steroids by impregnation with propylene glycol. A mixture of tritiated 5 alpha-androstan-3 alpha,17 beta-diol, testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3,17-dione was applied to the plate and developed in a toluene mobile phase to a height of 13.6 cm. This resulted in complete resolution of the 4 compounds as detected by a gas flow scanner or imaging analyzer. Cutting and elution of peak areas with methanol resulted in quantitative recovery of all four steroids. The thinness of the layer also permitted a 3-5% counting efficiency on scanning, resulting in good quantitation of recovery without liquid scintillation counting. The high sorptive capacity of the polyamide layer also enabled extracts of normal human serum to be defatted on the TLC plate by development with pure hexane prior to the toluene step. The new method thus offers several advantages over existing methods for steroid separations and should be adaptable to separations of other relatively non-polar compounds.     

A biuret method for determination of protein in hyamine hydroxide and NCS solutions used for liquid scintillation counting in toluene

A procedure for measurement of protein concentration in Hyamine hydroxide or NCS solution is described. The assay is linear from 0.5 to 8.0 mg. Use of this procedure offers the advantage of allowing liquid scintillation counting in toluene and protein assay on aliquots of the same sample.     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]-mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.     

Dissolved Hydrocarbons and Related Microflora in a Fjordal Seaport: Sources, Sinks, Concentrations, and Kinetics

The continuous addition of toluene as a solute of treated ballast water from oil tankers into a well-defined estuary facilitated the study of the dynamics of dissolved hydrocarbon metabolism in seawater. Most rates of toluene oxidation were in the range of 1 to 30 pg/liter per h at 0.5 μg of toluene per liter. Near the ballast water injection point, a layer of warm ballast water, rich in bacteria, that was trapped below the less-dense fresh surface water was located. Toluene residence times were approximately 2 weeks in this layer, 2 years elsewhere in Port Valdez, and 2 decades in the surface water of a more oceanic receiving estuary adjacent. Mixing was adequate for a steady-state treatment which showed that 98% of the toluene was flushed from Port Valdez before metabolism and gave a steady-state concentration of 0.18 μg/liter. Total bacterial biomass from direct counts and organism size data was usually near 0.1 mg/liter, but ranged up to 0.8 mg/liter in the bacteria-rich layer. The origin of bacteria in this layer was traced to growth in oil tanker ballast during shipments. The biomass of toluene oxidizers in water samples was estimated from the average affinity of pure-culture isolates for toluene (28 liters per g of cells per h) and observed toluene oxidation kinetics. Values ranged from nearly all of the total bacterial biomass within the bacteria-rich layer down to 0.2% at points far removed. Because the population of toluene oxidizers was large with respect to the amount of toluene consumed and because water from a nearby nonpolluted estuary was equally active in facilitating toluene metabolism, we searched for an additional hydrocarbon source. It was found that terpenes could be washed from spruce trees by simulated rainfall, which suggested that riparian conifers provide an additional and significant hydrocarbon source to seawater.     

A radiometric assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O₂ oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N-(5′-phosphopyridoxyl)[³H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting.     

Liquid scintillation counting of radioactive monomeric and polymeric carbohydrates on paper chromatograms

A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol.The water elution-Aquasol procedure allows water-soluble substances (¹⁴C- and ³H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.     

Diazomethane: improved analytical and distillation techniques for small quantities and some synthetic applications

An improved design of apparatus for the small-scale (about 5 μmol to about 50 mmol) preparation of diazomethane is described. The diazomethane is generated from commonly used precursors and distilled by aeration in a glass apparatus connected by Teflon tubing and without a condenser. A new simple and reasonably accurate procedure for assay of diazomethane is described. This depends on reaction with excess [¹⁴C]benzoic acid in toluene followed by quantitative removal of the excess acid by partitioning with pH 10 aqueous buffer and assaying the methyl [¹⁴C]benzoate in the toluene by liquid scintillation counting. Examples are given of the use of accurately known amounts of diazomethane and [¹⁴C] diazomethane for the preparation of methylated derivatives of [2-¹⁴C]barbital, 4′-hydroxy-[2-¹⁴C]phenobarbital, and mephobarbital. Small amount(s) of dimethyl-barbital (O-methyl) were separated and partly characterized by gas chromatography/mass spectroscopy and NMR.     

A simple method for the liquid scintillation counting of precipitated protein from red blood cells

A method for the liquid scintillation counting of precipitated protein from red cells in 0.1–1.0 ml of blood is described. Precipitate is incubated for 0.5 hr at 100°C with equal volumes of acetic acid, ethyl acetate, and hydrogen peroxide; an equal volume of hydrochlorie acid is then added, followed by a toluene/Triton X-100 scintillation mixture containing primary and secondary scintillators. Maximum counting efficiencies with precipitate from 0.2 ml of blood were 90% for ¹⁴C and 35% for ³H. Recovery of labeled amino acid was not less than 90%. Chemiluminescence decayed to not more than 15 cpm above background in 45 min.     

Investigations into the growth behaviour of a toluene-degrading Pseudomonas strain,taking the high vapour pressure of toluene into consideration

A toluene-degrading Pseudomonas species isolated from waste water was studied with regard to its growth behaviour. The dependence of the growth rate on the pH value, on the toluene concentration and on temperature, as well as the O₂ consumption of the isolate were determined. The fact that toluene is a highly volatile substrate, which is present both in the liquid and in the has phase of a reactor, was considered in the experiments. It is shown that the volatility of toluene has to be taken into consideration for an accurate determination of the toluene concentration in the medium.     

Toluene biodegradation rates in unsaturated soil systems versus liquid batches and their relevance to field conditions

Contaminant biodegradation in unsaturated soils may reduce the risks of vapor intrusion. However, the reported rates show large variability and are often derived from slurry experiments that are not representative of unsaturated conditions. Here, different laboratory setups are used to derive the biodegradation capacity of an unsaturated soil layer through which gaseous toluene migrates from the water table upwards. Experiments in static unsaturated soil microcosms at 6–30 % water-filled porosity (WFP) and unsaturated soil columns at 9, 14, and 27 % WFP were compared with liquid batches containing the same culture of Alicycliphilus denitrificans. The biodegradation rates for the liquid batches were orders of magnitude lower than for the other setups. Hence, liquid batches do not necessarily reflect optimal conditions for bacteria; either oxygen or toluene mass transfer at the cell scale or the absence of soil–water–air interfaces seemed to be limiting bacterial activity. For the column setup, the rates were limited by mass supply. The microcosm results could be described by apparent first-order biodegradation constants that increased with WFP or through a numerical model that included biodegradation as a first-order process taking place in the liquid phase only. The model liquid phase first-order rates varied between 6.25 and 20 h^?1 and were not related to the water content. Substrate availability was the primary factor limiting bioactivity, with evidence for physiological stress at the lowest water-filled porosity. The presented approach is useful to derive liquid phase biodegradation rates from experimental data and to include biodegradation in vapor intrusion models.     

An intact assay for enzymes that labilize C--H bonds.

The activity of enzymes that labilize known CH bonds can be estimated in intact tissue by following the loss to water of tritium from specifically labeled substrates. The accumulated tritium oxide (³HHO) in the medium is recovered by direct sublimation of aliquots of the medium into scintillation vials and the radio-activity measured by liquid scintillation spectrometry.