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Enzymatic method for measuring the absolute value of oxygen concentration
ANALYTICAL
BIOCHEMISTRY
179,124-126
(1989)
Enzymatic Method for Measuring the Absolute Value of Oxygen Concentration Tetsuya Yomo, Itaru Urabe, and Hirosuke Okada Department
of Fermentation
Technology,
Faculty of Engineering,
Osaka University
ReceivedSeptember16,1988
An enzymatic method for measuring the absolute concentration of oxygen in aqueous solutions, using 4-hydroxybenzoate 3-monooxygenase and glucose oxidase, is described. The monooxygenase is used for quantitative oxidation of 4-hydroxybenzoate and NADPH with oxygen into 3,4-dihydroxybenzoate and NADP+; the amount of oxygen can be measured as the amount of NADPH decreased by the reaction. The monooxygenase reaction is performed in a syringe, a closed system. After the completion of the monooxygenase reaction, glucose oxidase is added to the assay solution to consume the oxygen from the atmosphere; this enables us to measure the NADPH concentration in the solution spectrophotometrically in an open system and to check the anaerobicity of closed systems. The oxygen concentrations at 25W of oxygen-saturated and air-saturated water were 1.10 and 0.23 mM, respectively. The value for argon-bubbled water was zero within the experimental error; this justifies the assay system. Thus, it is shown that a sample containing 8 pM-1.1 mM oxygen can be measured by this method. o 1999 Academic PUBS, IUC.
Oxygen has various effects on living organisms, and it is important for biological and chemical researchers to measure oxygen concentrations in aqueous solutions accurately by a simple method. Several methods have been used, including chemical and electrode methods. For a chemical method, the Winkler titration method has been used widely to obtain absolute values because of its high sensitivity (l-3). This method, however, requires a large volume of sample solution and complex steps are necessary to remove from the sample solution various substances affecting the assay. Electrode methods, on the other hand, are convenient, but they give only relative values; that is, they require calibration (4-7), and the values are sensitive to temperature. Recently, Vanderkooi et al. have reported an optical method based on the oxygen-dependent quenching of phosphorescence (8), but this method also requires calibration. 124
The above drawbacks seem to be overcome by using enzyme reactions. Enzymes such as NADH oxidase systems have been used for calibration of oxygen (4-7), but enzymatic methods, in the true senseof the words, have not been developed for the assay of oxygen. In this work, we describe a new enzymatic method which gives oxygen concentration directly without calibration. In addition, this method does not require special instrumentation, and the enzymes used are commercially available. The main enzyme reaction used in our method is 4-hydroxybenzoate
+ O2 + NADPH
+ H+ + 3,4-dihydroxybenzoate
+ Hz0 + NADP+
4-Hydroxybenzoate 3-monooxygenase (EC 1.14.13.2) catalyzes this virtually irreversible reaction, and the amount of oxygen in a sample solution can be measured as the amount of NADPH decreased by the reaction. The most important point in measuring oxygen is to avoid the effects of atmospheric oxygen. In our method, the reaction of the monooxygenase is completed in a closed system within a short time (1 min), and then an excess of glucose oxidase (EC 1.1.3.4) over the monooxygenase is added to the assay mixture to consume the oxygen coming from the atmosphere instantaneously and completely. The presence of glucose oxidase and glucose in the assay solution enables us to measure the concentration of NADPH in the solution spectrophotometritally in an open system (under atmospheric oxygen). MATERIALS
AND METHODS
Materials
4-Hydroxybenzoate 3-monooxygenase from Pseudosp. and glucose oxidase from Aspergillus sp. were purchased from Toyobo Co. Ltd. (Osaka); sodium 4-hydroxybenzoate was from Nakarai Chemicals (Kyoto); NADPH was from Oriental Yeast Co. Ltd. (Tokyo). monas
0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMATIC
ASSAY
I 0
0.2
Fraction
0.4
0.6
0.6
of Oxygen-Saturated
1.0
of Enzyme
and Substrate
Solutions
4-Hydroxybenzoate 3-monooxygenase was dissolved in 0.1 M sodium phosphate, pH 7.5, at a concentration of 12.6 units/ml. The enzyme solution was warmed at 25°C and deaerated by bubbling argon gas. Glucose oxidase was dissolved in the same buffer at a concentration of 190 units/ml; this solution was kept at 0°C and also bubbled with argon gas. A solution containing 1.75 mM 4hydroxybenzoate, about 0.65 mM NADPH, 0.1 M glucose, and 0.1 M sodium phosphate, pH 7.5, was used as a substrate solution for the assay of oxygen. The substrate solution was warmed at 25°C and bubbled with argon gas. The concentration of NADPH in the substrate solution need not be adjusted accurately to 0.65 mM, because the actual value is measured later as the initial concentration of NADPH in the assay mixture as described below. Procedure
125
OXYGEN
tion was taken into the syringe, the reaction mixture was then transferred into a test tube (an open system), and the concentration of NADPH in the mixture was measured spectrophotometrically at 340 nm using a molar absorption coefficient of 6300 M-’ cm-’ with a Hitachi 220A spectrophotometer. All assays in this report were made in duplicate, and the difference in the absorbances between the duplicated values was within 5%. The initial concentration of NADPH in the assay mixture was measured as follows: 1 ml of water bubbled with argon, 2 ml of the substrate solution, and 1 ml of the glucose oxidase solution were taken into a syringe; after 1 min, the monooxygenase solution was taken into the syringe, and the concentration of NADPH was measured in the open system as described above. This NADPH concentration must be measured when the substrate solution is newly prepared.
Water
FIG. 1. Linear relationship between the oxygen concentration in sample solutions and the decrease in absorbance at 340 nm (&&) of the assay solution. Samples with different oxygen concentrations were prepared by mixing different volumes of oxygen-bubbled water and argon-bubbled water in a syringe (total 1 ml). The other procedures were the same as those described under Materials and Methods. The initial value of the absorbance was 1.534. The values of the absorbance after the assay procedures for the samples of the oxygen-bubbled water and the argon-bubbled water were 0.204 and 1.588, respectively.
Preparation
OF
of Oxygen Assay
One milliliter of the sample solution to be measured was taken into a syringe, any bubbles in the syringe were removed, and 2 ml of the substrate solution was taken into the syringe. The main reaction was started by taking 1 ml of 4-hydroxybenzoate 3-monooxygenase solution into the syringe (a closed system). One minute after the start of the reaction, 1 ml of the glucose oxidase solu-
RESULTS
AND
DISCUSSION
The value of the initial concentration of NADPH in the assay mixture prepared this time was 0.251 mM. This value did not change during the measurement of the absorbance at 340 nm in the open system described under Materials and Methods; this means that the reaction rate of glucose oxidase is much higher than that of the monooxygenase, and the oxygen diffusing from the atmosphere into the assay solution is completely consumed by the reaction of glucose oxidase. Thus, the addition of glucose oxidase after the completion of the monooxygenase reaction (this is the procedure for oxygen assay described in this report) eliminates the effects of atmospheric oxygen that cause overestimation of the oxygen concentration in a sample solution. If all the procedures are performed in a closed system, the addition of glucose oxidase may not be necessary. However, the addition of the oxidase ensures the anaerobic conditions; that is, this method can be used for checking the anaerobicity of closed systems. The oxygen concentration at 25°C of pure water bubbled with oxygen or air was measured by the procedures described under Materials and Methods, and the value was 1.10 or 0.23 mM, respectively; the ratio of these values (0.23/1.10 = 0.209) coincides well with the partial pressure of oxygen in the atmosphere. The value for the air-saturated water is 91% of that reported by Truesdale et al. (2) and 89% of that reported by Reynafarje et al. (7). The reason for these discrepancies is not clear, but generally, the oxygen concentration is apt to be overestimated due to the effects of atmospheric oxygen. In our method, the absence of such effects is confirmed as described above, but there is a possibility of underestimating oxygen concentrations if the reaction of the monooxygenase is not completed within 1 min. To check this point, the course of the decrease in absorbance at 340 nm was monitored just after the addition of the mono-
126
YOMO,
URABE,
oxygenase solution (1 ml) to the solution containing oxygen-bubbled water (1 ml), argon-bubbled water (1 ml), and the substrate solution (2 ml). The initial rapid decrease had finished abruptly, and the slope of the trace of the absorbance was the same as that for the reaction mixture containing the argon-bubbled water instead of the oxygen-bubbled water; even with a diluted monooxygenase solution (4.2 units/ml), the reaction had finished within 30 s. It is thus confirmed that the monooxygenase reaction is completed within 1 min under our assay conditions, and therefore, the values obtained by the enzymatic method seem to be correct. To check the linearity of our assay system, samples with different oxygen concentrations were prepared by taking into a syringe different volumes of the oxygenbubbled water and the argon-bubbled water (total 1 ml), and the decrease in absorbance at 340 nm due to the monooxygenase reaction was measured for each sample by the oxygen assay method described under Materials and Methods. The results shown in Fig. 1 indicate the accuracy and applicability of our method for the assay of a sample containing 8 pM-1.1 mM of oxygen. The upper detection limit depends on the NADPH concentration in the substrate solution, and it is possible to measure concentrations of oxygen higher than 1.1 mM. It should be pointed out that when the argon-bubbled water was used as a sample solution, the value of the NADPH concentration of the assay mixture was the same as that of the initial concentration of NADPH; this means that the value for oxygen-free water is measured to be zero within experimental error and shows the lower detection limit
AND
OKADA
of 8 PM, which corresponds the decrease in the absorbance of 0.01, the detection limit for the photometer. It is one of the merits of our method that the value for the argon-bubbled water can be measured, this value has been assumed to be zero for the other methods. This enables us to check the accuracy of the assay system. In summary, the enzymatic method described in this report has a high specificity to oxygen and gives reliable absolute values of oxygen concentration directly without any calibration. In principle, this method is applicable to a wide range of aqueous media unless they completely inhibit the enzymes or they change the concentration of NADPH under the assay conditions. In addition, this method does not require special instruments like electrodes, and the enzymes are commercially available. The principle of this method is also applicable to checking other systems for oxygen assay. REFERENCES 1. Winkler, L. W. (1889) Ber Dtsch. Chem. Ges. 22,1764-1774. 2. Truesdale, G. A., Downing, A. L., and Lowden, G. F. (1955) J. Appl. Chem. 5,53-62. 3. Montgomery, H. A. C., Thorn, N. S., and Cockburn, A. (1964) J. Appl. Chem. 14,280-296. 4. Chappell, J. B. (1964) 5. Hinkle, P. C., andYu,
Biochem. J. 90,225-237. M. L. (1979) J. Biol. Chem.
254,2450-2455.
6. Lemasters, J. J. (1984) J. BioE. Chcm. 269,13,123-13,130. 7. Reynafarje, B., Costa, L. E., and Lehninger, A. L. (1985) them. 145,406-418. 8. Vanderkooi, J. M., Maniara, G., Green, (1987) J. Biol. Chem. 262,5476-5482.
Ad.
T. J., and Wilson,
BioD. F.
BIOCHEMISTRY
179,124-126
(1989)
Enzymatic Method for Measuring the Absolute Value of Oxygen Concentration Tetsuya Yomo, Itaru Urabe, and Hirosuke Okada Department
of Fermentation
Technology,
Faculty of Engineering,
Osaka University
ReceivedSeptember16,1988
An enzymatic method for measuring the absolute concentration of oxygen in aqueous solutions, using 4-hydroxybenzoate 3-monooxygenase and glucose oxidase, is described. The monooxygenase is used for quantitative oxidation of 4-hydroxybenzoate and NADPH with oxygen into 3,4-dihydroxybenzoate and NADP+; the amount of oxygen can be measured as the amount of NADPH decreased by the reaction. The monooxygenase reaction is performed in a syringe, a closed system. After the completion of the monooxygenase reaction, glucose oxidase is added to the assay solution to consume the oxygen from the atmosphere; this enables us to measure the NADPH concentration in the solution spectrophotometrically in an open system and to check the anaerobicity of closed systems. The oxygen concentrations at 25W of oxygen-saturated and air-saturated water were 1.10 and 0.23 mM, respectively. The value for argon-bubbled water was zero within the experimental error; this justifies the assay system. Thus, it is shown that a sample containing 8 pM-1.1 mM oxygen can be measured by this method. o 1999 Academic PUBS, IUC.
Oxygen has various effects on living organisms, and it is important for biological and chemical researchers to measure oxygen concentrations in aqueous solutions accurately by a simple method. Several methods have been used, including chemical and electrode methods. For a chemical method, the Winkler titration method has been used widely to obtain absolute values because of its high sensitivity (l-3). This method, however, requires a large volume of sample solution and complex steps are necessary to remove from the sample solution various substances affecting the assay. Electrode methods, on the other hand, are convenient, but they give only relative values; that is, they require calibration (4-7), and the values are sensitive to temperature. Recently, Vanderkooi et al. have reported an optical method based on the oxygen-dependent quenching of phosphorescence (8), but this method also requires calibration. 124
The above drawbacks seem to be overcome by using enzyme reactions. Enzymes such as NADH oxidase systems have been used for calibration of oxygen (4-7), but enzymatic methods, in the true senseof the words, have not been developed for the assay of oxygen. In this work, we describe a new enzymatic method which gives oxygen concentration directly without calibration. In addition, this method does not require special instrumentation, and the enzymes used are commercially available. The main enzyme reaction used in our method is 4-hydroxybenzoate
+ O2 + NADPH
+ H+ + 3,4-dihydroxybenzoate
+ Hz0 + NADP+
4-Hydroxybenzoate 3-monooxygenase (EC 1.14.13.2) catalyzes this virtually irreversible reaction, and the amount of oxygen in a sample solution can be measured as the amount of NADPH decreased by the reaction. The most important point in measuring oxygen is to avoid the effects of atmospheric oxygen. In our method, the reaction of the monooxygenase is completed in a closed system within a short time (1 min), and then an excess of glucose oxidase (EC 1.1.3.4) over the monooxygenase is added to the assay mixture to consume the oxygen coming from the atmosphere instantaneously and completely. The presence of glucose oxidase and glucose in the assay solution enables us to measure the concentration of NADPH in the solution spectrophotometritally in an open system (under atmospheric oxygen). MATERIALS
AND METHODS
Materials
4-Hydroxybenzoate 3-monooxygenase from Pseudosp. and glucose oxidase from Aspergillus sp. were purchased from Toyobo Co. Ltd. (Osaka); sodium 4-hydroxybenzoate was from Nakarai Chemicals (Kyoto); NADPH was from Oriental Yeast Co. Ltd. (Tokyo). monas
0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMATIC
ASSAY
I 0
0.2
Fraction
0.4
0.6
0.6
of Oxygen-Saturated
1.0
of Enzyme
and Substrate
Solutions
4-Hydroxybenzoate 3-monooxygenase was dissolved in 0.1 M sodium phosphate, pH 7.5, at a concentration of 12.6 units/ml. The enzyme solution was warmed at 25°C and deaerated by bubbling argon gas. Glucose oxidase was dissolved in the same buffer at a concentration of 190 units/ml; this solution was kept at 0°C and also bubbled with argon gas. A solution containing 1.75 mM 4hydroxybenzoate, about 0.65 mM NADPH, 0.1 M glucose, and 0.1 M sodium phosphate, pH 7.5, was used as a substrate solution for the assay of oxygen. The substrate solution was warmed at 25°C and bubbled with argon gas. The concentration of NADPH in the substrate solution need not be adjusted accurately to 0.65 mM, because the actual value is measured later as the initial concentration of NADPH in the assay mixture as described below. Procedure
125
OXYGEN
tion was taken into the syringe, the reaction mixture was then transferred into a test tube (an open system), and the concentration of NADPH in the mixture was measured spectrophotometrically at 340 nm using a molar absorption coefficient of 6300 M-’ cm-’ with a Hitachi 220A spectrophotometer. All assays in this report were made in duplicate, and the difference in the absorbances between the duplicated values was within 5%. The initial concentration of NADPH in the assay mixture was measured as follows: 1 ml of water bubbled with argon, 2 ml of the substrate solution, and 1 ml of the glucose oxidase solution were taken into a syringe; after 1 min, the monooxygenase solution was taken into the syringe, and the concentration of NADPH was measured in the open system as described above. This NADPH concentration must be measured when the substrate solution is newly prepared.
Water
FIG. 1. Linear relationship between the oxygen concentration in sample solutions and the decrease in absorbance at 340 nm (&&) of the assay solution. Samples with different oxygen concentrations were prepared by mixing different volumes of oxygen-bubbled water and argon-bubbled water in a syringe (total 1 ml). The other procedures were the same as those described under Materials and Methods. The initial value of the absorbance was 1.534. The values of the absorbance after the assay procedures for the samples of the oxygen-bubbled water and the argon-bubbled water were 0.204 and 1.588, respectively.
Preparation
OF
of Oxygen Assay
One milliliter of the sample solution to be measured was taken into a syringe, any bubbles in the syringe were removed, and 2 ml of the substrate solution was taken into the syringe. The main reaction was started by taking 1 ml of 4-hydroxybenzoate 3-monooxygenase solution into the syringe (a closed system). One minute after the start of the reaction, 1 ml of the glucose oxidase solu-
RESULTS
AND
DISCUSSION
The value of the initial concentration of NADPH in the assay mixture prepared this time was 0.251 mM. This value did not change during the measurement of the absorbance at 340 nm in the open system described under Materials and Methods; this means that the reaction rate of glucose oxidase is much higher than that of the monooxygenase, and the oxygen diffusing from the atmosphere into the assay solution is completely consumed by the reaction of glucose oxidase. Thus, the addition of glucose oxidase after the completion of the monooxygenase reaction (this is the procedure for oxygen assay described in this report) eliminates the effects of atmospheric oxygen that cause overestimation of the oxygen concentration in a sample solution. If all the procedures are performed in a closed system, the addition of glucose oxidase may not be necessary. However, the addition of the oxidase ensures the anaerobic conditions; that is, this method can be used for checking the anaerobicity of closed systems. The oxygen concentration at 25°C of pure water bubbled with oxygen or air was measured by the procedures described under Materials and Methods, and the value was 1.10 or 0.23 mM, respectively; the ratio of these values (0.23/1.10 = 0.209) coincides well with the partial pressure of oxygen in the atmosphere. The value for the air-saturated water is 91% of that reported by Truesdale et al. (2) and 89% of that reported by Reynafarje et al. (7). The reason for these discrepancies is not clear, but generally, the oxygen concentration is apt to be overestimated due to the effects of atmospheric oxygen. In our method, the absence of such effects is confirmed as described above, but there is a possibility of underestimating oxygen concentrations if the reaction of the monooxygenase is not completed within 1 min. To check this point, the course of the decrease in absorbance at 340 nm was monitored just after the addition of the mono-
126
YOMO,
URABE,
oxygenase solution (1 ml) to the solution containing oxygen-bubbled water (1 ml), argon-bubbled water (1 ml), and the substrate solution (2 ml). The initial rapid decrease had finished abruptly, and the slope of the trace of the absorbance was the same as that for the reaction mixture containing the argon-bubbled water instead of the oxygen-bubbled water; even with a diluted monooxygenase solution (4.2 units/ml), the reaction had finished within 30 s. It is thus confirmed that the monooxygenase reaction is completed within 1 min under our assay conditions, and therefore, the values obtained by the enzymatic method seem to be correct. To check the linearity of our assay system, samples with different oxygen concentrations were prepared by taking into a syringe different volumes of the oxygenbubbled water and the argon-bubbled water (total 1 ml), and the decrease in absorbance at 340 nm due to the monooxygenase reaction was measured for each sample by the oxygen assay method described under Materials and Methods. The results shown in Fig. 1 indicate the accuracy and applicability of our method for the assay of a sample containing 8 pM-1.1 mM of oxygen. The upper detection limit depends on the NADPH concentration in the substrate solution, and it is possible to measure concentrations of oxygen higher than 1.1 mM. It should be pointed out that when the argon-bubbled water was used as a sample solution, the value of the NADPH concentration of the assay mixture was the same as that of the initial concentration of NADPH; this means that the value for oxygen-free water is measured to be zero within experimental error and shows the lower detection limit
AND
OKADA
of 8 PM, which corresponds the decrease in the absorbance of 0.01, the detection limit for the photometer. It is one of the merits of our method that the value for the argon-bubbled water can be measured, this value has been assumed to be zero for the other methods. This enables us to check the accuracy of the assay system. In summary, the enzymatic method described in this report has a high specificity to oxygen and gives reliable absolute values of oxygen concentration directly without any calibration. In principle, this method is applicable to a wide range of aqueous media unless they completely inhibit the enzymes or they change the concentration of NADPH under the assay conditions. In addition, this method does not require special instruments like electrodes, and the enzymes are commercially available. The principle of this method is also applicable to checking other systems for oxygen assay. REFERENCES 1. Winkler, L. W. (1889) Ber Dtsch. Chem. Ges. 22,1764-1774. 2. Truesdale, G. A., Downing, A. L., and Lowden, G. F. (1955) J. Appl. Chem. 5,53-62. 3. Montgomery, H. A. C., Thorn, N. S., and Cockburn, A. (1964) J. Appl. Chem. 14,280-296. 4. Chappell, J. B. (1964) 5. Hinkle, P. C., andYu,
Biochem. J. 90,225-237. M. L. (1979) J. Biol. Chem.
254,2450-2455.
6. Lemasters, J. J. (1984) J. BioE. Chcm. 269,13,123-13,130. 7. Reynafarje, B., Costa, L. E., and Lehninger, A. L. (1985) them. 145,406-418. 8. Vanderkooi, J. M., Maniara, G., Green, (1987) J. Biol. Chem. 262,5476-5482.
Ad.
T. J., and Wilson,
BioD. F.