Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

Immobilization of luciferase from a firefly lantern extract on glass strips as an alternative strategy for luminescent detection of ATP

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L -lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at −80°C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research. © 1998 John Wiley & Sons, Ltd.     

Cell-surface-localized ATP detection with immobilized firefly luciferase

We demonstrate the application of an assay technique for the detection of ATP efflux from the cell surface. Until recently, the firefly luciferase assay has conventionally been used to detect ATP release because of its high sensitivity and wide detection ability. However, since this technique can be used only to infer the amount of diffused ATP in bulk solutions, it does not accurately reflect the dynamic ATP flux at the cell membrane. We therefore constructed luciferase fusion proteins that could be immobilized onto the cell surface. However, the low activities and limited application ranges of these proteins prompted us to use biotinylated luciferase given its attributes of strong and stable localization. Furthermore, luciferase can be immobilized strongly onto the biotinylated cell surface via streptavidin-biotin binding and can thus be used to determine the dynamic release of ATP near the cell surface.     

ATP生物发光测定试剂研究进展

萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能。迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等。ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面。     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Effect of solvents on the catalytic activity of firefly luciferase

Various solvents stimulate the catalytic activity of firefly luciferase, up to sevenfold. Polyvinylpyrrolidone, polyethylene glycols, and nonionic detergents such as Triton X-100 were the most effective stimulators of the enzyme. Both peak light and total light emission were enhanced in the presence of these solvents indicating an increased turnover of the enzyme. The primary effect of the solvents is on the oxidative reaction rather than the activation reaction. All the experimental data support the hypothesis that the presence of solvent promotes the dissociation of the inhibitory product from the enzyme.     

Automated bioluminescent assays for NADH, glucose 6-phosphate, primary bile acids, and ATP

An automated flow system for the bioluminescent assay of various metabolites have been developed. The enzymes used in the assays have been coimmobilized onto Sepharose and packed into small flow cells. Assays for NADH, glucose 6-phosphate, and primary bile acids utilize the bacterial NADH:FMN oxidoreductase/luciferase and either glucose-6-phosphate dehydrogenase or 7 alpha-hydroxysteroid dehydrogenase. ATP assays were performed using immobilized firefly luciferase. In general, the lower limit of detection of the metabolites was at the picomole level, and light intensity was proportional to the substrate concentration from several picomoles to several hundred picomoles. The reproducibility was good with coefficient of variations in the range of 2-5%. The carryover was less than 5% and 30 samples per hour could be assayed. The flow cells were reusable for up to 700 consecutive assays. The major factor limiting their continued use was bacterial contamination of the Sepharose. The results obtained for serum primary bile acids using the bioluminescent assay wer in good agreement with independent measurements on the same samples using gas-liquid chromatography. The immobilized firefly luciferase system was successfully used to measure high levels of bacteria in urine specimens.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Determination of adenosine 5'-triphosphate by the luciferase system with a stopped-flow spectrometer.

A stopped-flow spectrometer is used for ATP assay by firefly luciferase-luciferin method. It allows one to record initial rise of the light intensity and to differentiate the light produced due to the conversion of ADP to ATP by nucleoside diphosphokinase in the firefly lantern when other nucleoside triphosphates are present. Addition of luciferin (0.27 mm) to luciferase extract increases the light intensity by a factor of 50–100. This method can be used to measure ATP in the picomole range.     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells.

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.     

A luminometric method for the determination of ATP and phosphocreatine in single human skeletal muscle fibres

A sensitive method for the analysis of ATP and phosphocreatine (PCr) in single human skeletal muscle fibres is described. Muscle tissue was freeze-dried and single fibres were dissected free with the aid of low-power microscopy. The fibres were then extracted in trichloroacetic acid and neutralized with KHCO₃. The assay is based on the continuous monitoring of light produced as a result of ATP degradation in the firefly luciferase reaction. PCr is measured as the amount of ATP formed in the creatine kinase reaction. The coefficient of variation was less than 4% for both ATP and PCr determination. The amount of tissue required for the assay is approximately 0.5 μg (dry weight). The assay showed good agreement with spectrophotometric and high-performance liquid chromatographic (HPLC) measurements made upon extracts of whole muscle tissue.     

Method for real-time detection of inorganic pyrophosphatase activity

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.     

Use of Firefly Luciferase for ATP Measurement: Other Nucleotides Enhance Turnover

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (>8μM) or a fairly constant production of light with lower concentrations of ATP (< 1 μM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

Factors affecting the kinetics of light emission from crude and purified firefly luciferase.

Crude and purified firefly luciferase have been used to assay ATP from 0.2 pmol to 2 μmol. Over this range of ATP concentrations, there is a large change in the kinetics of light emission. At the lowest concentrations of ATP, light emission rises to a maximum and remains constant for a minute or longer. As the concentration of ATP is increased, the peak light intensity increases and the decay rate of light increases significantly. This is true for both the crude as well as the purified enzyme. High concentration of sodium arsenate as well as other salts inhibit the peak light emission and prevent the decay in light intensity which is due to product inhibition. It is possible to obtain almost any type of kinetics by manipulating the experimental conditions.     

Bioluminescent methods of analysis in microbiology

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.