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A new fluorimetric method for measurement of oxidase-catalyzed reactions
Analytica Chimica Acta. 117 (1980) 363-365 o Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
Short Communication
A NEW FLUORIMETRIC OXIDASE-CATALYZED
LINDA B. McGOWN*, Department (U.S.A.)
of Chemistry,
METHOD FOR MEASUREMENT REACTIONS
BRADLEY California
J. KIRST and GARY State
University.
OF
L. LaROWE
Long
Beach,
California
90840
(Received 15th January 1980) Summary. The oxidase-catalyzed oxidation of glucose consumes dissolved oxygen, the rate of disappearance of which is monitored by oxygen quenching of the fluorescence of pyrene in a 1 :l waterethanol medium. The relationship between the rate of increase of fluorescence and concentration is linear for 10 pg-1 mg of glucose in the optical cell.
The quenching of fluorescence by dissolved osygen is generally regarded as a nuisance, necessitating the removal of the oxygen prior to measuring fluorescence emission. However, the quenching effect can also be a valuable analytical tool, for example in the determination of the oxygen content of solutions [ 11 . A general method based on the quenching of fluorescence by oxygen is proposed here for measurement of oxidase systems. As the oxidase reaction proceeds, dissolved oxygen is consumed. If a fluorescent species which is susceptible to quenching by oxygen is present in the solution, its emission intensity will increase during the course of the oxidase-catalyzed reaction. The successful application of this system to measurement of the glucose oxidase-catalyzed oxidation of glucose is described. With appropriate modification, the method should be generally applicable to any osidase system.
Experimental Apparatus.
All fluorescence measurements were made on an AmincoBowman Spectrofluorometer, using a IO-mm quartz cell. All oxygen electrode measurements were made on a Beckman Glucose Analyzer_ Reagents_ For the pyrene solution (2.4 X lo3 M), dissolve 4.84 mg of pyrene in 1 1 of absolute ethanol (reagent quality)_ Prepare a glucose oxidase solution (100 I.U. ml-‘) by adding 1 ml of glucose osidase (Sigma, Cat. No. G-6500, 1400 I.U. ml-‘) to 13 ml of water, and store at 4°C. Prepare glucose solutions by adding 200 mg of p-D-glucose to 100 ml of water, and store at 4°C to prevent bacterial growth. Use deionized water throughout. Procedure. Add 0.250 ml of pyrene solution and 0.250 ml of glucose oxidase solution to 2.00 ml of 1:l water-ethanol solution in a sample cuvet. Mix thoroughly. Add 0.150 ml of glucose sample, invert the cuvet once, and immediately place in the spectrofluorometer cell compartment. Monitor the
361
= 359 nm) on the recorder and measure of the reaction (Fig. 1). Plot the maxi-
emission intensity (ACx = 334 nm, A,, the maximum rate (masimum slope)
mum matesagainst known glucose ~on~en~rat~ons in order to obtain a calibration graph _
Reszrfts and discrtssion Glucose osidase
was chosen for initial study and demonstration of the fluorescence quenching method because of the wide availability of the inespensive enzyme and substrate preparations. Also, the behavior of the enzyme has been weil studied, and areas of appfication for glucose assays are widespread. including food, clinical, and biochemical sciences. Choice of fluorescent ~~i~~~~i~~d and soloetzt. The molecule used as the
fluorescent indicator has to be susceptible to osygen quenching, and show a measurable change in the estent of quenching for the range of dissolved oxygen variation produced by the osidase-catalyzed reaction_ The molecule must, also be soluble in a solution which will allow appreciable activity of the enzyme, i.e. an aqueous system. X number of water-soluble molecules were tried, all having fluorescent life-times on the order of 1O-8-1O-g s, including fluorescein, 2,7-dichlorofluoresccin, salicylic acid and quinine. Although they ~$1 showed susceptibility to osygen quenching when measured in oxygenated solutions, none showed sufficient sensitivity for monitoring the glucose osidase reaction, Pyrcne was tried because of its longer fl uorexence iifetime (1 WY s), which would make it more susceptible to quenching by oxygen. Indeed, it proved to be suitable for monitoring the oxidase reaction_ Pyrene in aqueous solution was found to have X,, (mas) at 334 nm and A,, (max) at 389nm, ascompared to reported values of 330 nm and 382 nm in pentane 121. Fluorescence escitation and emission spectra for pyrene were identical for pyrene alone, pyrene and glucose, and pyrene and glucose osidase. The spectra before and after de-oxygenation showed an increase in fluorescence intensity in the absence of oxygen, but no change in the wavelength distribution of the spectra, indicating that the quenching is not due to a change in the structure of pyrene.
I i
5cr --... 20
Cl W
,A_
.+ /
L -I
I-
O
f.._._-_-..
IO
19 i,P.Si
-
/
90
Fig. 1. Changes in pyrene ffuorescence intensity (F.I.) during a giucose c&&se reaction (upper traces) immediately after addition of glucose. The numbers above the cwves are mg of glucose added. The straight lines and numbers show the maximum slopes for each curve and their retative numerical values.
365
Pyrene is soluble in ethanol but insoluble in water, so it is necessary to use a solution of ethanol in water to monitor osidase reactions by using pyrene. By observing the intensity of the scattering peak (X,, = X,, = 389 nm) in the fluorescence emission spectrum, it, was found that pyrene (2.26 X 10m6 kl in the cell) is soluble in solutions containing at least 30% ethanol. Below 30% ethanol, scattering intensity begins to increase, because of precipitation. A 50% ethanol solution was chosen for subsequent measurements to insure that no precipitation would occur. Amperometric measurement of the rate of oxygen consumption with an oxygen sensor was used to observe the effect of the solvent system on the glucose osidase reaction rate. There was a 7370 increase in the reaction rate in 1:l water-, ethanol compared with a wholly aqueous solution_ A 10% decrease in rate was observed when pyrene (2.26 X lo-’ &I) was miscd with the 1:l water-ethanol solution, relative to the rate without pyrene. An increase in pyrene concentration produced an increased fluorescence intensity but did not increase the sensitivity of the osidase measurements, i.e. the rate of change of intensity remained constant. Effect of glucose nnd enzyme concentrations. ‘The reaction rate increased linearly with glucose osidase concentration up to 27 1-U. in the cell; rates could be measured for concentrations as low as 2.7 1-U. in the cell. There was a linear relationship between glucose concentration (O-l -00 mg of glucose in the cell) and the rate of intensity increase. As little as 10 Dg of glucose could be measured. The coefficient of variation for 13 determinations on a 2.01 g 1-l glucose solution (0.50 mg in the cell) was 9%. Conclusions. The use of oxygen quenching to monitor oxidase systems, as described above for glucose osidase, has proved to be an effective approach. The method is rapid, selective and simple, and would not be subject to interference from fluorescent components of biological samples, because of the kinetic nature of the measurements. Precision and sensitivity could be improved by using more sophisticated sample addition and mising techniques, as well as by using more direct, electronic means of finding the maximum reaction rate. REFERENCES 1 E. Sawicki, T. Hawser and T. Stanley, Int. J. Air Pollut., 2 (1960) 253. 2 C. A. Parker, Photoluminescence of Solutions, Elsevier, Amsterdam, 195.
1968, pp. 493-
Printed in The Netherlands
Short Communication
A NEW FLUORIMETRIC OXIDASE-CATALYZED
LINDA B. McGOWN*, Department (U.S.A.)
of Chemistry,
METHOD FOR MEASUREMENT REACTIONS
BRADLEY California
J. KIRST and GARY State
University.
OF
L. LaROWE
Long
Beach,
California
90840
(Received 15th January 1980) Summary. The oxidase-catalyzed oxidation of glucose consumes dissolved oxygen, the rate of disappearance of which is monitored by oxygen quenching of the fluorescence of pyrene in a 1 :l waterethanol medium. The relationship between the rate of increase of fluorescence and concentration is linear for 10 pg-1 mg of glucose in the optical cell.
The quenching of fluorescence by dissolved osygen is generally regarded as a nuisance, necessitating the removal of the oxygen prior to measuring fluorescence emission. However, the quenching effect can also be a valuable analytical tool, for example in the determination of the oxygen content of solutions [ 11 . A general method based on the quenching of fluorescence by oxygen is proposed here for measurement of oxidase systems. As the oxidase reaction proceeds, dissolved oxygen is consumed. If a fluorescent species which is susceptible to quenching by oxygen is present in the solution, its emission intensity will increase during the course of the oxidase-catalyzed reaction. The successful application of this system to measurement of the glucose oxidase-catalyzed oxidation of glucose is described. With appropriate modification, the method should be generally applicable to any osidase system.
Experimental Apparatus.
All fluorescence measurements were made on an AmincoBowman Spectrofluorometer, using a IO-mm quartz cell. All oxygen electrode measurements were made on a Beckman Glucose Analyzer_ Reagents_ For the pyrene solution (2.4 X lo3 M), dissolve 4.84 mg of pyrene in 1 1 of absolute ethanol (reagent quality)_ Prepare a glucose oxidase solution (100 I.U. ml-‘) by adding 1 ml of glucose osidase (Sigma, Cat. No. G-6500, 1400 I.U. ml-‘) to 13 ml of water, and store at 4°C. Prepare glucose solutions by adding 200 mg of p-D-glucose to 100 ml of water, and store at 4°C to prevent bacterial growth. Use deionized water throughout. Procedure. Add 0.250 ml of pyrene solution and 0.250 ml of glucose oxidase solution to 2.00 ml of 1:l water-ethanol solution in a sample cuvet. Mix thoroughly. Add 0.150 ml of glucose sample, invert the cuvet once, and immediately place in the spectrofluorometer cell compartment. Monitor the
361
= 359 nm) on the recorder and measure of the reaction (Fig. 1). Plot the maxi-
emission intensity (ACx = 334 nm, A,, the maximum rate (masimum slope)
mum matesagainst known glucose ~on~en~rat~ons in order to obtain a calibration graph _
Reszrfts and discrtssion Glucose osidase
was chosen for initial study and demonstration of the fluorescence quenching method because of the wide availability of the inespensive enzyme and substrate preparations. Also, the behavior of the enzyme has been weil studied, and areas of appfication for glucose assays are widespread. including food, clinical, and biochemical sciences. Choice of fluorescent ~~i~~~~i~~d and soloetzt. The molecule used as the
fluorescent indicator has to be susceptible to osygen quenching, and show a measurable change in the estent of quenching for the range of dissolved oxygen variation produced by the osidase-catalyzed reaction_ The molecule must, also be soluble in a solution which will allow appreciable activity of the enzyme, i.e. an aqueous system. X number of water-soluble molecules were tried, all having fluorescent life-times on the order of 1O-8-1O-g s, including fluorescein, 2,7-dichlorofluoresccin, salicylic acid and quinine. Although they ~$1 showed susceptibility to osygen quenching when measured in oxygenated solutions, none showed sufficient sensitivity for monitoring the glucose osidase reaction, Pyrcne was tried because of its longer fl uorexence iifetime (1 WY s), which would make it more susceptible to quenching by oxygen. Indeed, it proved to be suitable for monitoring the oxidase reaction_ Pyrene in aqueous solution was found to have X,, (mas) at 334 nm and A,, (max) at 389nm, ascompared to reported values of 330 nm and 382 nm in pentane 121. Fluorescence escitation and emission spectra for pyrene were identical for pyrene alone, pyrene and glucose, and pyrene and glucose osidase. The spectra before and after de-oxygenation showed an increase in fluorescence intensity in the absence of oxygen, but no change in the wavelength distribution of the spectra, indicating that the quenching is not due to a change in the structure of pyrene.
I i
5cr --... 20
Cl W
,A_
.+ /
L -I
I-
O
f.._._-_-..
IO
19 i,P.Si
-
/
90
Fig. 1. Changes in pyrene ffuorescence intensity (F.I.) during a giucose c&&se reaction (upper traces) immediately after addition of glucose. The numbers above the cwves are mg of glucose added. The straight lines and numbers show the maximum slopes for each curve and their retative numerical values.
365
Pyrene is soluble in ethanol but insoluble in water, so it is necessary to use a solution of ethanol in water to monitor osidase reactions by using pyrene. By observing the intensity of the scattering peak (X,, = X,, = 389 nm) in the fluorescence emission spectrum, it, was found that pyrene (2.26 X 10m6 kl in the cell) is soluble in solutions containing at least 30% ethanol. Below 30% ethanol, scattering intensity begins to increase, because of precipitation. A 50% ethanol solution was chosen for subsequent measurements to insure that no precipitation would occur. Amperometric measurement of the rate of oxygen consumption with an oxygen sensor was used to observe the effect of the solvent system on the glucose osidase reaction rate. There was a 7370 increase in the reaction rate in 1:l water-, ethanol compared with a wholly aqueous solution_ A 10% decrease in rate was observed when pyrene (2.26 X lo-’ &I) was miscd with the 1:l water-ethanol solution, relative to the rate without pyrene. An increase in pyrene concentration produced an increased fluorescence intensity but did not increase the sensitivity of the osidase measurements, i.e. the rate of change of intensity remained constant. Effect of glucose nnd enzyme concentrations. ‘The reaction rate increased linearly with glucose osidase concentration up to 27 1-U. in the cell; rates could be measured for concentrations as low as 2.7 1-U. in the cell. There was a linear relationship between glucose concentration (O-l -00 mg of glucose in the cell) and the rate of intensity increase. As little as 10 Dg of glucose could be measured. The coefficient of variation for 13 determinations on a 2.01 g 1-l glucose solution (0.50 mg in the cell) was 9%. Conclusions. The use of oxygen quenching to monitor oxidase systems, as described above for glucose osidase, has proved to be an effective approach. The method is rapid, selective and simple, and would not be subject to interference from fluorescent components of biological samples, because of the kinetic nature of the measurements. Precision and sensitivity could be improved by using more sophisticated sample addition and mising techniques, as well as by using more direct, electronic means of finding the maximum reaction rate. REFERENCES 1 E. Sawicki, T. Hawser and T. Stanley, Int. J. Air Pollut., 2 (1960) 253. 2 C. A. Parker, Photoluminescence of Solutions, Elsevier, Amsterdam, 195.
1968, pp. 493-