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3-(p-Hydroxyphenyl)propionic acid as a new fluorogenic reagent for amine oxidase assays
ANALYTICAL
BIOCHEMISTRY
138,
133-136
(1984)
3-(p-Hydroxyphenyl)propionic Acid as a New Fluorogenic Reagent for Amine Oxidase Assays TARATOSHI
MATSUMOTO, *J TAMAKI FURUTA,* YUJI NIMURA,* AND OSAMU SUZUKI?
*Division of Oncology, First Department of Surgery, Nagoya University School of Medicine, Nagoya 466, and tDepartment of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan Received August 17, 1983 A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(phydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.
For assaysof oxidases, the method originally described by Guilbault et al. (1) and Snyder and Hendley (2) has been widely used. In this method, hydrogen peroxide formed in the oxidase reaction is measured fluorometrically by converting homovanillic acid (HVA)’ to a fluorescent compound in the presence of peroxidase. Recently, Zaitsu and Ohkura (3) have tested a number of substrates other than HVA, and recommended 3-(p-hydroxyphenyl) propionic acid (HPPA) as the best substrate for peroxidase in its sensitivity and stability. In the present paper, we have applied this peroxidase-HPPA system to assays for amine oxidases and found that the alkalinization of the medium after enzyme reaction results in a three- to fivefold increase in its fluorescence; thus we could develop an extremely sensitive assay method for amine oxidases. MATERIALS
AND
METHODS
Chemicals. HPPA was purchased from Dojin Company, Ltd., Kumamoto, Japan; horseradish peroxidase (type I), HVA, pu’ To whom all inquiries should be addressed. * Abbreviations used: HPPA, 3-(phydroxyphenyl)propionic acid; HVA, homovanillic acid; MAO, monoamine oxidase; DAO, diamine oxidase; PAO, polyamine oxidase.
trescine-2HC1, pargyline-HCI, and hog kidney diamine oxidase (DAO) were from the Sigma Chemical Company, St. Louis, Missouri; tyramine-HCl was from Nakarai Chemicals, Ltd., Kyoto; semicarbazide-HCl was from Kanto Chemical Company, Inc., Tokyo; and hydrogen peroxide was from Mitsubishi-Gasukagaku, Ltd., Tokyo. Other common chemicals used were of the highest purity commercially available. Monoamine oxidase (MAO). As MAO preparations, crude mitochondrial fractions were isolated from various organs of male Sprague-DawIey rats and humans that had died of head injury, pneumonia, apoplexy, liver cirrhosis, and heart failure, as described previously (4). Protein determinations. Enzyme protein was measured by a modification (5) of the conventional biuret method. Apparatus. The fluorescence intensity and spectra were measured in a Hitachi 650-10s fluorescence spectrophotometer at 25°C. The pH’s of the mixtures were measured with a Hitachi-Horiba type F-7 pH meter. Efect of alkalinization. Hydrogen peroxide (2.2 nmol) was reacted with HPPA and peroxidase to produce a fluorescent compound, to which 2.0 ml of water or 2.0 ml of 0.1 N NaOH solution was added. It was found that 133
0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
134
MATSUMOTO
the alkalinization after the enzyme reaction markedly enhanced the fluorescence. Figure 1 shows excitation and emission spectra of the fluorescent product with and without the alkalinization. The excitation spectrum without alkalinization showed three peaks, at 250,295, and 320 nm (Fig. lA), while that with alkalinization showed two peaks, at 263 and 320 nm (Fig. 1B). Both emission spectra showed similar patterns with peaks at 4 10 nm. Figure 2 shows fluorescence intensities as a function of the final pH values of the reaction media in the assays of MAO, DAO, and hydrogen peroxide itself with the peroxidaseHPPA system, where enzymatic reactions have been carried out at pH 7.4. The fluorescence of the test samples was equally increased remarkably from pH 6.5 to pH 10.0, and it remained at high levels up to pH 12.5 and then declined. The fluorescence of the blank tests was also increased up to pH 10.0 and then decreased. Since the sensitivity of fluorometric assays greatly depends on the ratio of fluorescence of a test sample to that of a blank test, the most desirable pH of the mixture was found to be around pH 12.0. The shift of pH of the mixture to this pH resulted in a three- to fivefold increase in fluorescence as compared with that measured at pH 7.4.
AL.
The alkalinization also contributed to the stopping of the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations such as crude homogenates. The fluorescence in the alkaline mixture was stable for several hours. Reaction conditions. The effect of the amount of HPPA in the assay mixture was examined with hydrogen peroxide, MAO, and DA0 as shown in Fig. 3. The optimal amount of HPPA was 0.1 mg/tube (incubation mixture, 0.6 ml; final volume, 2.6 ml). The effect of peroxidase concentration was also tested, as shown in Fig. 4. For purified DA0 and hydrogen peroxide itself, the fluorescence intensity did not change appreciably over the wide range of the peroxidase concentrations, but for crude rat liver mitochondrial MAO it was highest at 1 purpurogallin unit peroxidase/tube, which was adopted in our assay system. Recommended procedure. On the basis of the above data, we recommend the following procedure as a standard assay for an amine oxidase or hydrogen peroxide itself. The incubation mixtures (0.6 ml) contain 0.10 ml of 0.5 M potassium phosphate buffer (pH 7.4) 0.10 ml of peroxidase solution (0.1 mgjml in B
A Emission 410
600
ET
500
400
Excitation
Emission
320 ,295
300
200 600 500 Wavelength (nm)
Excitation
410
400
320
300
200
FIG. 1. Excitation and emission spectra of a fluorescent compound produced by the reaction of peroxidase with HPPA and hydrogen peroxide, with (B) and without (A) alkalinization after the enzyme reaction. The lower curves show the respective blank fluorescence. The fluorescence was monitored at 410 nm for the excitation spectra, and the excitation at 320 nm was used for the emission spectra. Spectral bandwidths were 5 nm. The amount of hydrogen peroxide added to the mixture was 2.2 nmol/tube (final volume, 2.6 ml).
NEW FLUOROMETRIC
ASSAY FOR AMINE
135
OXIDASES
0 0.1
0.01 0
2
4
6
8
10
12
14
PH
FIG 2. Effect of final pH values of reaction mixtures on the fluorescence produced by the reaction of rat liver mitochondrial MAO (0) hog kidney DA0 (A), or hydrogen peroxide (0). The upper curves show the fluorescence of test samples and the lower curves that of their respective biank tests. The amounts of MAO, DAO, and hydrogen peroxide were 8.6 fig protein, 40 pg solid, and 2.2 nmol/tube, respectively. The concentration of the substrates, tyramine for MAO and putrescine for DAO, was 1.OmM. The reaction of MAO and DA0 was stopped by adding 0.2 mM pargyline and 3.0 mM semicarbazide, respectively, prior to the addition of 2.0 ml of NaOH or HCl solutions of various concentrations. For details of the assays,see the text. Each point represents the mean of duplicate determinations.
Peroxidase
10
1 (purpurogallin
unit/tube)
FIG. 4. Effect of peroxidase concentrations on the tluorescence produced by the reaction of rat liver mitochondrial MAO (0) hog kidney DA0 (A), or hydrogen peroxide (0). The assayconditions are the same as specified in Fig. 2, except that the enzyme reaction was stopped only by adding 2.0 ml of 0.1 N NaOH solution.
case of 100 purpurogallin units/mg solid), 0.10 ml of HPPA solution (1 mg/ml), 0.1 ml of test solution (MAO, DAO, or hydrogen peroxide), 0.10 ml of substrate solution (tyramine for MAO, or putrescine for DAO), and 0.10 ml of water. They are incubated at 37°C for an appropriate period ( 15-60 min), after which 2.0 ml of 0.1 N NaOH is added to the mixture to bring its final pH to around pH 12.0. The fluorescence intensity is measured with excitation at 320 nm and emission at 4 10 nm (uncorrected). As blank tests, assay mixtures without substrates are incubated; the substrates are mixed after adding the NaOH solution. Internal standards should be taken by adding appropriate amounts (0.1-2.2 nmol) of hydrogen peroxide to the mixtures prior to incubation, especially when some quenching due to enzyme preparations is suspected. RESULTS
0.001
0.01
0.1
1.0
HPPA (mg/tube)
FIG. 3. Effect of HPPA concentrations on the fluorescence produced by the reaction of rat liver mitochondrial MAO (O), hog kidney DA0 (A), or hydrogen peroxide (0). The assay conditions are the same as specified in Fig. 2, except that the enzyme reaction was stopped only by adding 2.0 ml of 0.1 N NaOH solution.
Reliability of the method. The calibration curve with various amounts of hydrogen peroxide, constructed by the present assay method, is presented in Fig. 5. Linearity could be obtained over the range 0.02-2.20 nmol/ tube. The detection limit was 0.02 nmol, which gave 150-200% of the blank fluorescence.
MATSUMOTO
136
The linearity was also checked with human kidney crude mitochondrial MAO with 1 mM tyramine as substrate, and also with hog kidney DA0 with 1 mM putrescine; the assays were linear up to 60 min of incubation time and up to 0.5 mg protein/tube of enzyme concentration. Table 1 shows MAO activities in crude mitochondrial preparations from various human organs measured with both HPPA and HVA; in the latter case the HPPA solution (1 mg/ ml) was replaced by a HVA solution (1 mg/ ml) (6). The values with HPPA generally agreed with those with HVA, supporting the validity of the present assay method. This was also true for the hog kidney DAO.
ET AL. TABLE 1 MAO ACTIVITIES
INTHE CRUDE MITCEHONDRIA F-ROMVARIOUSHUMANORGANSMEASURED WITHHPPAANDHVA
MAO activity toward tyramine (1 .O mM)” (nmol H202 formed mg protein-’ 30 mini’) Organ
HPPA
Liver Kidney Heart Small intestine Lung Pancreas Spleen
207 ?I 21 203 f 29 96.9 zk 6.4 90.7 k 39.7 35.0 * 9.1 10.3 +- 7.9 1.8 f 0.7
HVA 220 191 103 95.4 37.5 10.7 5.0
f 17 +- 18 + 7 k 34.4 f 9.2 f 6.2 f 0.9
n Means f SE, obtained from four to five experiments, are given.
DISCUSSION
In the present study, we have employed HPPA for assays of MAO and DAO, and also have found that alkalinization of the reaction medium results in a remarkable enhancement of its fluorescence intensity. The fluorescence intensity obtained in our method was about 8 times higher than that in the original HVA method (2) and 2.2 times higher than that in our modified HVA method (6) (unpublished observation). In addition, the blank levels in the present assay were much lower than those in the HVA methods (2,6) (unpublished observation). Therefore, this method is so sen-
sitive that as little as 0.02 nmol hydrogen peroxide can be measured accurately (Fig. 5). We have also tested HPPA for the assays of polyamine oxidase (PAO) in human kidney mitochondria with N’-monoacetylspermine as substrate and compared it with the HVA method; the PA0 activity with HPPA was found to be less than 50% of that with HVA, probably due to inhibition of PA0 by HPPA (unpublished observation). Thus we cannot recommend the use of HPPA for assays of mammalian PA0 at the present time. Since our method needs neither extraction nor centrifugation, it is so simple and rapid that more than 100 samples can be treated within 3 h. This HPPA method with alkalinization will also be very useful for the assays of other oxidases producing hydrogen peroxide. REFERENCES 1. Guilbault, G. G., Brignac, P. J., Jr., and Juneau, M. (1968) Anal. Chem. 40, 1256-1263. 2. Snyder, S. H., and Hendley, E. D. ( 1968) J Pharmacol. Exp.
I
1
0
0.5
1.0 Hz02
/
I
1.5
2.0
2.5
(nmoles/tube)
FIG. 5. Fluorescence intensity as a function of hydrogen peroxide concentrations. Each point represents the mean obtained from duplicate determinations.
Ther.
163, 386-392.
3. Zaitsu, K., and Ohkura, Y. (1980) Anal. Biochem. 109, 109-l 13. 4. Suzuki, O., Katsumata, Y., Oya, M., and Matsumoto, T. (1979) B&hem. Pharmacol. 28, 2327-2332. 5. Suzuki, O., Noguchi, E., and Yagi, K. (1977) Brain Rex 135, 305-313. 6. Matsumoto, T., Furuta, T., Nimura, Y., and Suzuki, 0. (1982) Biochem. Pharmacol. 31, 2207-2209.
BIOCHEMISTRY
138,
133-136
(1984)
3-(p-Hydroxyphenyl)propionic Acid as a New Fluorogenic Reagent for Amine Oxidase Assays TARATOSHI
MATSUMOTO, *J TAMAKI FURUTA,* YUJI NIMURA,* AND OSAMU SUZUKI?
*Division of Oncology, First Department of Surgery, Nagoya University School of Medicine, Nagoya 466, and tDepartment of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan Received August 17, 1983 A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(phydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.
For assaysof oxidases, the method originally described by Guilbault et al. (1) and Snyder and Hendley (2) has been widely used. In this method, hydrogen peroxide formed in the oxidase reaction is measured fluorometrically by converting homovanillic acid (HVA)’ to a fluorescent compound in the presence of peroxidase. Recently, Zaitsu and Ohkura (3) have tested a number of substrates other than HVA, and recommended 3-(p-hydroxyphenyl) propionic acid (HPPA) as the best substrate for peroxidase in its sensitivity and stability. In the present paper, we have applied this peroxidase-HPPA system to assays for amine oxidases and found that the alkalinization of the medium after enzyme reaction results in a three- to fivefold increase in its fluorescence; thus we could develop an extremely sensitive assay method for amine oxidases. MATERIALS
AND
METHODS
Chemicals. HPPA was purchased from Dojin Company, Ltd., Kumamoto, Japan; horseradish peroxidase (type I), HVA, pu’ To whom all inquiries should be addressed. * Abbreviations used: HPPA, 3-(phydroxyphenyl)propionic acid; HVA, homovanillic acid; MAO, monoamine oxidase; DAO, diamine oxidase; PAO, polyamine oxidase.
trescine-2HC1, pargyline-HCI, and hog kidney diamine oxidase (DAO) were from the Sigma Chemical Company, St. Louis, Missouri; tyramine-HCl was from Nakarai Chemicals, Ltd., Kyoto; semicarbazide-HCl was from Kanto Chemical Company, Inc., Tokyo; and hydrogen peroxide was from Mitsubishi-Gasukagaku, Ltd., Tokyo. Other common chemicals used were of the highest purity commercially available. Monoamine oxidase (MAO). As MAO preparations, crude mitochondrial fractions were isolated from various organs of male Sprague-DawIey rats and humans that had died of head injury, pneumonia, apoplexy, liver cirrhosis, and heart failure, as described previously (4). Protein determinations. Enzyme protein was measured by a modification (5) of the conventional biuret method. Apparatus. The fluorescence intensity and spectra were measured in a Hitachi 650-10s fluorescence spectrophotometer at 25°C. The pH’s of the mixtures were measured with a Hitachi-Horiba type F-7 pH meter. Efect of alkalinization. Hydrogen peroxide (2.2 nmol) was reacted with HPPA and peroxidase to produce a fluorescent compound, to which 2.0 ml of water or 2.0 ml of 0.1 N NaOH solution was added. It was found that 133
0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
134
MATSUMOTO
the alkalinization after the enzyme reaction markedly enhanced the fluorescence. Figure 1 shows excitation and emission spectra of the fluorescent product with and without the alkalinization. The excitation spectrum without alkalinization showed three peaks, at 250,295, and 320 nm (Fig. lA), while that with alkalinization showed two peaks, at 263 and 320 nm (Fig. 1B). Both emission spectra showed similar patterns with peaks at 4 10 nm. Figure 2 shows fluorescence intensities as a function of the final pH values of the reaction media in the assays of MAO, DAO, and hydrogen peroxide itself with the peroxidaseHPPA system, where enzymatic reactions have been carried out at pH 7.4. The fluorescence of the test samples was equally increased remarkably from pH 6.5 to pH 10.0, and it remained at high levels up to pH 12.5 and then declined. The fluorescence of the blank tests was also increased up to pH 10.0 and then decreased. Since the sensitivity of fluorometric assays greatly depends on the ratio of fluorescence of a test sample to that of a blank test, the most desirable pH of the mixture was found to be around pH 12.0. The shift of pH of the mixture to this pH resulted in a three- to fivefold increase in fluorescence as compared with that measured at pH 7.4.
AL.
The alkalinization also contributed to the stopping of the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations such as crude homogenates. The fluorescence in the alkaline mixture was stable for several hours. Reaction conditions. The effect of the amount of HPPA in the assay mixture was examined with hydrogen peroxide, MAO, and DA0 as shown in Fig. 3. The optimal amount of HPPA was 0.1 mg/tube (incubation mixture, 0.6 ml; final volume, 2.6 ml). The effect of peroxidase concentration was also tested, as shown in Fig. 4. For purified DA0 and hydrogen peroxide itself, the fluorescence intensity did not change appreciably over the wide range of the peroxidase concentrations, but for crude rat liver mitochondrial MAO it was highest at 1 purpurogallin unit peroxidase/tube, which was adopted in our assay system. Recommended procedure. On the basis of the above data, we recommend the following procedure as a standard assay for an amine oxidase or hydrogen peroxide itself. The incubation mixtures (0.6 ml) contain 0.10 ml of 0.5 M potassium phosphate buffer (pH 7.4) 0.10 ml of peroxidase solution (0.1 mgjml in B
A Emission 410
600
ET
500
400
Excitation
Emission
320 ,295
300
200 600 500 Wavelength (nm)
Excitation
410
400
320
300
200
FIG. 1. Excitation and emission spectra of a fluorescent compound produced by the reaction of peroxidase with HPPA and hydrogen peroxide, with (B) and without (A) alkalinization after the enzyme reaction. The lower curves show the respective blank fluorescence. The fluorescence was monitored at 410 nm for the excitation spectra, and the excitation at 320 nm was used for the emission spectra. Spectral bandwidths were 5 nm. The amount of hydrogen peroxide added to the mixture was 2.2 nmol/tube (final volume, 2.6 ml).
NEW FLUOROMETRIC
ASSAY FOR AMINE
135
OXIDASES
0 0.1
0.01 0
2
4
6
8
10
12
14
PH
FIG 2. Effect of final pH values of reaction mixtures on the fluorescence produced by the reaction of rat liver mitochondrial MAO (0) hog kidney DA0 (A), or hydrogen peroxide (0). The upper curves show the fluorescence of test samples and the lower curves that of their respective biank tests. The amounts of MAO, DAO, and hydrogen peroxide were 8.6 fig protein, 40 pg solid, and 2.2 nmol/tube, respectively. The concentration of the substrates, tyramine for MAO and putrescine for DAO, was 1.OmM. The reaction of MAO and DA0 was stopped by adding 0.2 mM pargyline and 3.0 mM semicarbazide, respectively, prior to the addition of 2.0 ml of NaOH or HCl solutions of various concentrations. For details of the assays,see the text. Each point represents the mean of duplicate determinations.
Peroxidase
10
1 (purpurogallin
unit/tube)
FIG. 4. Effect of peroxidase concentrations on the tluorescence produced by the reaction of rat liver mitochondrial MAO (0) hog kidney DA0 (A), or hydrogen peroxide (0). The assayconditions are the same as specified in Fig. 2, except that the enzyme reaction was stopped only by adding 2.0 ml of 0.1 N NaOH solution.
case of 100 purpurogallin units/mg solid), 0.10 ml of HPPA solution (1 mg/ml), 0.1 ml of test solution (MAO, DAO, or hydrogen peroxide), 0.10 ml of substrate solution (tyramine for MAO, or putrescine for DAO), and 0.10 ml of water. They are incubated at 37°C for an appropriate period ( 15-60 min), after which 2.0 ml of 0.1 N NaOH is added to the mixture to bring its final pH to around pH 12.0. The fluorescence intensity is measured with excitation at 320 nm and emission at 4 10 nm (uncorrected). As blank tests, assay mixtures without substrates are incubated; the substrates are mixed after adding the NaOH solution. Internal standards should be taken by adding appropriate amounts (0.1-2.2 nmol) of hydrogen peroxide to the mixtures prior to incubation, especially when some quenching due to enzyme preparations is suspected. RESULTS
0.001
0.01
0.1
1.0
HPPA (mg/tube)
FIG. 3. Effect of HPPA concentrations on the fluorescence produced by the reaction of rat liver mitochondrial MAO (O), hog kidney DA0 (A), or hydrogen peroxide (0). The assay conditions are the same as specified in Fig. 2, except that the enzyme reaction was stopped only by adding 2.0 ml of 0.1 N NaOH solution.
Reliability of the method. The calibration curve with various amounts of hydrogen peroxide, constructed by the present assay method, is presented in Fig. 5. Linearity could be obtained over the range 0.02-2.20 nmol/ tube. The detection limit was 0.02 nmol, which gave 150-200% of the blank fluorescence.
MATSUMOTO
136
The linearity was also checked with human kidney crude mitochondrial MAO with 1 mM tyramine as substrate, and also with hog kidney DA0 with 1 mM putrescine; the assays were linear up to 60 min of incubation time and up to 0.5 mg protein/tube of enzyme concentration. Table 1 shows MAO activities in crude mitochondrial preparations from various human organs measured with both HPPA and HVA; in the latter case the HPPA solution (1 mg/ ml) was replaced by a HVA solution (1 mg/ ml) (6). The values with HPPA generally agreed with those with HVA, supporting the validity of the present assay method. This was also true for the hog kidney DAO.
ET AL. TABLE 1 MAO ACTIVITIES
INTHE CRUDE MITCEHONDRIA F-ROMVARIOUSHUMANORGANSMEASURED WITHHPPAANDHVA
MAO activity toward tyramine (1 .O mM)” (nmol H202 formed mg protein-’ 30 mini’) Organ
HPPA
Liver Kidney Heart Small intestine Lung Pancreas Spleen
207 ?I 21 203 f 29 96.9 zk 6.4 90.7 k 39.7 35.0 * 9.1 10.3 +- 7.9 1.8 f 0.7
HVA 220 191 103 95.4 37.5 10.7 5.0
f 17 +- 18 + 7 k 34.4 f 9.2 f 6.2 f 0.9
n Means f SE, obtained from four to five experiments, are given.
DISCUSSION
In the present study, we have employed HPPA for assays of MAO and DAO, and also have found that alkalinization of the reaction medium results in a remarkable enhancement of its fluorescence intensity. The fluorescence intensity obtained in our method was about 8 times higher than that in the original HVA method (2) and 2.2 times higher than that in our modified HVA method (6) (unpublished observation). In addition, the blank levels in the present assay were much lower than those in the HVA methods (2,6) (unpublished observation). Therefore, this method is so sen-
sitive that as little as 0.02 nmol hydrogen peroxide can be measured accurately (Fig. 5). We have also tested HPPA for the assays of polyamine oxidase (PAO) in human kidney mitochondria with N’-monoacetylspermine as substrate and compared it with the HVA method; the PA0 activity with HPPA was found to be less than 50% of that with HVA, probably due to inhibition of PA0 by HPPA (unpublished observation). Thus we cannot recommend the use of HPPA for assays of mammalian PA0 at the present time. Since our method needs neither extraction nor centrifugation, it is so simple and rapid that more than 100 samples can be treated within 3 h. This HPPA method with alkalinization will also be very useful for the assays of other oxidases producing hydrogen peroxide. REFERENCES 1. Guilbault, G. G., Brignac, P. J., Jr., and Juneau, M. (1968) Anal. Chem. 40, 1256-1263. 2. Snyder, S. H., and Hendley, E. D. ( 1968) J Pharmacol. Exp.
I
1
0
0.5
1.0 Hz02
/
I
1.5
2.0
2.5
(nmoles/tube)
FIG. 5. Fluorescence intensity as a function of hydrogen peroxide concentrations. Each point represents the mean obtained from duplicate determinations.
Ther.
163, 386-392.
3. Zaitsu, K., and Ohkura, Y. (1980) Anal. Biochem. 109, 109-l 13. 4. Suzuki, O., Katsumata, Y., Oya, M., and Matsumoto, T. (1979) B&hem. Pharmacol. 28, 2327-2332. 5. Suzuki, O., Noguchi, E., and Yagi, K. (1977) Brain Rex 135, 305-313. 6. Matsumoto, T., Furuta, T., Nimura, Y., and Suzuki, 0. (1982) Biochem. Pharmacol. 31, 2207-2209.