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European J Appl Microbiol Biotechnol (1982) 15: 258-264

Microbiologyand Notechn 9

gy

S p r i n g e r - V e r l a g - 1982

Continuous Flow Method for Extraction and Bioluminescence Assay of ATP in Baker's Yeast Maj-Rita Siro 1 , Henrik Romar 1 , and Timo L6vgren 2 1 Department of Biochemistry and Pharmacy, Abo Akademi SF-20500 Turku 50, Finland 2 WallacBiochemical Laboratory, P.O. Box 10, SF-20101 Turku 10, Finland

Summary. The intracellular ATP of baker's yeast (Saccharomyces cerevisiae) was measured using the bioluminescent firefly luciferase assay. Benzalkonium chloride and trichloro-acetic acid served in the experiments as extracting agents and optimal conditions for the extraction and assay of the intracellular ATP are reported. Using the results obtained from manually performed experiments two continuous flow systems were designed for the measurement of ATP in yeast cells during cell growth. Good correlation between the amount of cellular ATP and cell growth was found during the exponential growth phase.

development of methods for effective and rapid extraction of ATP from the micro-organisms. The extraction of ATP from yeast cells has, unlike somatic cells, proved to be quite difficult because of their resistant cell envelopes. The present study was undertaken in order to develop an extraction procedure o f yeast ATP that is easy to perform, results in a high yield and is well suited to the bioluminescence assay. Another purpose of the work was to develop a continuous flow system designed for measurement of yeast cell ATP which could serve as an index of yeast culture growth.

Materials and Methods

Introduction In the study of cell metabolism the assay of the adenine nucleotides is of interest and many methods have been developed for their determination 0tolm-Hansen and Karl 1978). The firefly luciferin-luciferase system described by McElroy (1963) has provided the basis for a specific and sensitive assay for the estimation of adenosine-5'triphosphate (ATP) in different biological materials. Sensitivity and specificity of the assay were considerably increased when purified luciferase was used instead of crude enzyme extracts (Lundin and Thore 1975a; Lundin et al. 1976). Microbiological applications of the bioluminescence assay comprise estimations of biomass and investigations of the metabolic state of cells. The principle has been applied, for example, to estimate the bacterial content in clinical specimes (Thore et al. 1975; Alexander et al. 1976), foods (Sharpe et al. 1970) and drinking water (Levin et al. 1968). The use of the bioluminescence assay in connection with bacteriological work prompted the Offprint requests to: Maj-Rita Siro 0171-1741/82/0015/0258/$ 01.40

Yeast. Commercial baker's yeast (Saccharomyces cerevisiae) propagated on molasses was obtained from Oy Alko Ab (Rajam~iki, Finland): Growth Conditions. To start the incubation, which was performed at room temperature without aeration, 1.5 g fresh yeast/l medium was used (Siro and L6vgren 1979). Growth was monitored by measuring absorbance at 554 nm. A linear relationship in absorbance to grams of yeast per liter was obtained. The number of cells per unit volume of the suspension of non-growing yeast was calculated using a BiJrker chamber. The value obtained was 7.6 x 106 cells/mg yeast. Assay ofA TP. The firefly-luciferase system was used to determine the concentration of cellular ATP. The method is highly specific (McEkoy and Green 1956) and based on the measurement of light emission produced during the oxidation of lucfferin by molecular oxygen in the presence of ATP and magnesium ions. The light intensity is directly proportional to the concentration of ATP (Lundin et al. 1976). The LKB-WallacLuminometer 1250 equipped with a potentiometrie recorder was used to record the light intensity. Measurements of the manually performed experiments were carried out in disposable polystyrene cuvettes and microlitre pi-

M.-R. Siro et al.: Continuous Bioluminescent Assay of ATP in Yeast

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pettes with disposable plastic tips were used in all determinations. The lucfferin-luciferase reagent (1243-200 ATP Monitoring Reagent, LKB-Wallac, Finland) was reconstituted in 10 ml distilled water. According to the manufacturer the linear range of the ATP assay is 10 -11 to 10. 6 M (Lundin and Thore 1975a). The assay mixture contained in order of addition, 0.1 M Tris-acetate buffer containing 2 mM EDTA, pH 7.75, 100 #1 of the ATP Monitoring Reagent and 10 ~1 of the diluted trichloro-acetic acid (TCA) extract or 25 #1 of the benzalkonium (BA) extract. The total volume of the assay mixture was 500 ~1. The assay was calibrated using an internal ATP standard of known concentration. This was performed by adding 5 ~1 10 - s M ATP Standard (1243-201 ATP Standard, LKB-Wallac, Finland) to the assay sample. The ATP level in the extract was calculated after correcting for the reagent background by using the signal produced by the ATP Standard as a reference. The Continuous Flow System. The flow system designed for the bioluminescent measurement of yeast cell ATP consisted of two major components, the AKEA Automatic Chemical Analysis System (Instrumentarium Oy, Finland) and the LKB-WalIac 1250 Luminometer with recorder. The pump had a constant speed of rotation and the amount of fluid pumped through each line was proportional to the inside cross-sectional area of the line. The flow in the lines was segmented by a bubble unit feeding air bubbles into the stream at regular intervals. A glass coil served as a flow cell in the luminometer.

Results

The first series of experiments was performed manually in order to find the most effective ATP extraction conditions and to establish the optimal assay conditions for the bioluminescence measurements. A number o f ionic and other compounds have been reported to interfere with the firefly-luciferase reaction (Holmsen et al. 1966; Rasmussen and Nielsen 1968; Lin and Cohen 1968; Thore 1979) and therefore the analytical interference of the extraction reagents used, TCA and BA, with the luciferase assay was first studied. The experiments were performed

b y adding 5 #1 of varying concentrations of extractant to the assay mixture containing 10 . 7 M ATP Standard. The emission of light in the presence and absence of extractant was compared. The output of Iight remained constant for several minutes for a given amount o f ATP in the absence o f the extractant and the analytical interference caused by the extractant was measured as changes in the light emission. Figure 1 shows the reduction in light emission as percentage inhibition and the decay rate o f the light o u t p u t as percentage decay per minute as a function o f the concentration of the extractant in the cuvette. The results clearly demonstrate that the inhibition of the luciferase assay is proportional to the amount of TCA in the cuvette. A concentration of 0.2% (w/v) TCA causes an inhibition of 50%. At the TCA concentrations used the decay of light emission per minute was less than 1%. The results also show that the concentration of BA in the assay mixture must be lower than 0.005% (w/v) in order to avoid a rapid decay of the light due to continuous inactivation of the luciferase enzyme. On the basis of the results obtained the ATP assay extraction conditions were chosen so that inhibition of the luciferase reaction was at an acceptable low level and the light output was stable. Before ATP measurement, the TCA extracts were diluted with 0.1 M Tris-acetate buffer containing 2 mM ethylenediaminetetra-acetic acid (EDTA), pH 7.75 to a final concentration of 0.007% (w/v) TCA in the assay mixture in the cuvette. The amount o f BA in the cuvette was 0.004% (w/v). The degree of inhibition of the luciferase reaction caused by TCA or BA in the extracts was

1.5%. The efficiency of varying concentrations of TCA and BA as extractants was studied and extractions on several different dilutions of the yeast suspension were performed in order to ensure that the yield of ATP was linearly related to the amount of sample. In manually performed experiments the extraction mixture contained either 100 #1 of the yeast suspension and 1 ml of a solution of TCA or

260

M.-R. Siro et al.: Continuous Bioluminescent Assay of ATP in Yeast

Table 1. The amounts of intracellular ATP extracted manually from varying amounts of yeast at different concentrations of BA in the extraction mixture BA in the extraction mixture % (w/v)

ATP (#tool x 10-4) extracted from the different amounts of yeast 5 #g

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Yeast, mg

100/al of the yeast suspension and 100 pl of a solution of BA. In the extraction solutions 4 mM EDTA was included. The approximate extraction time was 1 min, during which time the mixtures were rotated using a Vortex mixer at room temperature. It was found that a 10% (w/v) solution of TCA, which equals 9% (w/v) TCA in the extraction mixture, resulted in the most effective extraction over a short time and allowed an acceptable dilution of the extract before the assay of ATP. The effect of different amounts of BA in the extraction mixture is shown in Table 1. BA concentrations (w/v) of 0.12%, 0.08%, 0.04% and 0.02% seemed to be equally effective at extracting ATP from the various amounts of yeast used. Linearity was obtained for the extraction using the 10% (w/v) TCA solution up to about 12 mg yeast (Fig. 2), this corresponding to 8 mg TCA/mg yeast. Using the 0.16% solution of BA, which equals the extraction concentration of 0.08%, lineafity was obtained up to 0.4 mg yeast this corresponding to 0.4 mg BA/mg yeast. More than 0.4 mg yeast resulted in unstable extracts with decreasing outputs of light. At 0.4 mg the decay of light emission per minute was 1%-2%. Extracts prepared with TCA were free of enzyme activities interfering with the assay. The yield of ATP per milligram of

yeast under the conditions described was 2 x 10 - 6 mmol and the same with BA and TCA. The optimal assayconditions of the flow system were established by the results obtained in the previous experiments. The flow diagram of the system using 10% (w/v) TCA as extractant is shown in Fig. 3. The corresponding system for 0.16% (w/v) BA differs only in the volume of the lines of the sample, the ATP extractant and the degree of dilution of the extract. In BA system the flow rate of th e sample and BA solution lines were 160/zl/min and that of the diluting buffer line 320 gl/min. In both systems the sample was injected into the segmented stream of extractant and the extraction of ATP from the cells took place in the mixing coil of appropriate length. The ATP Monitoring Reagent was pumped into the system immediately before the diluted sample entered the luminometer. Intermittent reagent injection into the line was carried out and the reagent was kept on ice. A standard solution of 10- 6 M ATP in buffer alternated with buffer in the line next to the ATP Monitoring Reagent. This arrangement allowed internal calibration of each measurement. The extraction solutions were 10% TCA (w/v) containing 4 mM EDTA or 0.16% BA (w/v) containing 4 mM EDTA, respectively. The response time, i.e, the time it takes to

M.-R. Siro et al.: Continuous Bioluminescent Assay of ATP in Yeast

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