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A fluoristatic method for the determination of alkaline phosphatase, sorbitol dehydrogenase, peroxidase and pseudocholinesterase
Anatyiica C~imictz Acta, 129 (1981) 231-236 ESevier Scientific Publishing Company, Amsterdam -Printed
in The Netberiands
Short Communication
A FLUORISTATIC METEIOD FOR THE DETEiRMINATION OF ALKALINE PHOSPHATASE, SORBITOL DEHYDROGENASE, PEROXIDASE AND PSEUDOCHOLIJY-ESTERASE
SIE;GBE;RT PANTEL
Lehrstuht fiir Analytische Chemie. Freiburg i.Br_ (W. Germany) (Received 25th
Chemisches
Laboratorium
der Uniuersit%,
January 1981)
Instrumentation for fluoristatic procedures and several applications are described. The simple device is used to determine alkaline phosphatase (7-150 mlJ), sorbitol dehydrogenase (l-10 z&J) and peroxidase (0.5-5 mu) with fluorescent substrates and pseudocbolinest (10-100 mu) with a non-fluorescent substrate and erythrosine as fluorescence quencher_ The method is of spec*& interest for the determination of enzymes with low substrate concentrations.
Summary_
The applicability of ‘%&at” methods in analyticaI chemistry for the determination of catalysts, activators and inhibitors has been reported quite recently [I--3] _ The ma& advantage of these open-system methods lies in the fact that well-defined, low concentrations of substrate or product can be kept constant for a much longer time than in closed systems. This is of special interest in enzymatic analysis, as has been pointed out earlier 111. Fluorimetry can be up to several orders of magnitude more sensitive than absorption spectrophotometry [4] _ It seemed therefore to be of interest to study the use of fluorescence for regulating the addition of a suitable reagent in a stat method. With the fluorist&ic technique described here, it is possible to determine inorganic catalysts and enzymes which react with a fluorescent substrate to form non-fluorescent products. This is done by controhed addition of the fluorescent substrate solution at a speed such that the fluorescence remains constant during the reaction time observed. The added volume per time unit is a measure of the catalyst concentration, the activity of the enzyme being given in units analogous to international units (1 I.U. = catalytic activity which transforms 1 JHIIO!of substrate per minute under the reaction conditions used). The technique can also be used to follow reactions that produce fluorescent products from non-fluorescent substrates. In this case, the increasing fluorescence is quenched by controlled addition of a suitable substance [5,6] at a speed such that again the fluorescence remains constant during the measuring time. This mode of working is not that of a conventional stat method; the substrate or product concentration is not kept constant during the reaction time, but a physicat property of the product is 0003-2670/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
diminished to a preset level. This method has the advantage that nonfluorescent substrates can be used for the determination of enzymes, and the disadvantage that the addition curves for the quenching substance are not linear over a long period of time, because the quenching effect is not linear with the added volume of the quenching solution. Some examples are given below for the determination of enzymes by means of fluorescent substrate. An example of the determination of an enzyme with a non-fIuorescent substrate producing a fluorescent reaction product is also described. Instrumentaticm The apparatus used (Fig. 1) consists of the Combi-Tifmtor 3 D (Metrohm, Herisau, Switzerland) [‘7] and the fluorescence unit of the MPI-System (McKee-Pedersen, Danville, USA). Procedure with a fluorescent substrate. Into the quartz cuvette (18 mm inner diameter) of the measuring compartment M (Fig. 1) a~ pippetted 2 ml of buffer sclution and water up to 7.5 ml. The well-stirred solution is thermostatted (circulating thermostat) for 5 min to 25.0 + O.I”C and the millivoltmeter is set to zero with the aid of the mV some (TJ), Now a definite volume of the appropriate titrant, equivalent to the substrate concentration to be kept constant in the enzyme reaction, is added to the cuvette from the l-ml burette of the Microdosigraph. For this titrant concentration, the millivoltmeter is set to 60 mV (most sensitive switching region; preset working potential) by means of the variable feed-back resistor Rf. After the insfxument has been set thus, the enzyme activity can be determined as described below, Enzyme and substrate solutions were stored at 0°C in the dark. Unless otherwise stated, enzymes and reagents were obtained from Bee_bringer, Mannheim.
Fig. 1. Schematic representation of the measuring/regulating circuit. M, Sample/fiIter holder SIP-1017 with magnetic stirrer NIP-1024; OA, Operational amplifier MP-1006; U, millivolt source MF-1008; 50-2 V; Ph, photomultiplier MP-1021; L, mercury lamp Hanau St. 41/220 V (Quarzlarnpen GmbH-Hanau, FRG) with stabilized voltage supply; mV, mV-meter E510 (Metrohm); Imp, irnpulsornac E473 (Metrohm); Bur./R.ec, microdosimat (l-ml burette) with recorder attached [Mekohm); F,, F,, optical interference fitters.
233 Determination of alkaline phosphatase with I-naphthylphosphate Alkaline phosphatase catalyzes the hydrolysis of l-naphthylphosphate
to form 1-naphthol [ 8]_ Both the substrate and the Ijroduct are fluorescent, but under suitable conditions (F, = 313 nm, F2 = 365 nm; Fig. l), the substrate can be measured selectively. Procedure. To the quartz cuvette are added 2 ml of sodium hydroxideglpcine buffer solution (pH 9.8, 0.2 M) and 0.05-l ml of a solution of alkaline phosphatase (EC 3.1.3.1, 0.15 U ml-‘) for preparing a calibration graph or a definite amount of the sample solution within the specified range. After dilution with water to 7.5 ml, this solution is thermostatted for 5 min. The millivoltmeter is set to zero and the titrant addition is started; an aqueous solution of monosodium-1-naphthylphosphate monohydrate (0.25 mg ml-l) (EGA, Steinheim) is used. The working potential (60 mV; Rf = 9 kohm) is chosen so as to maintain a constant substrate concentration of 25 PM 1-naphthylphosphate in the solution. A typical recorder plot is given in Fig. 2. The calibration graph (tan Q versus enzyme concentration) is slightly and smoothly curved towards the tan Q axis, but passes through the origin of the coordinates. Table 1 gives some results for the determination of alkaline phosphatase. Determination of sorbitol dehydrogenase adenine dinucleotide (NADH)
with
reduced
p-nicotinamide
Sorbitol dehydrogenase (SDH) catalyzes the reduction of D-fructose with NADH [I, 9, lo], which is fluorescent (F, = 365 nm, F, = 458 nm). The resulting NAD+ does not fluoresce under these conditions. Procedure. To the quartz cuvette are added 1 ml of sodium phosphate buffer solution (pH 6.1, 0.3 M), 2 ml of D-fructose solution (10 mg ml-‘) and 0.1-l ml of a solution of SDH (EC 1.1.1.14, 10 mU ml-l) for preparing a calibration graph or an appropriate amount of the sample solution_ After dilution with water to 7.5 ml, this solution is thermostatted for 5 min, the millivoltmeter is set to zero and addition of the titrant (0.1 mg of p-NADHNa2 per ml of water) is started. The working potential (60 mV;
, 3 min.
Fig. 2. Recordergraphfor the determination of 90 mU of alkalinephosphatase in 7.5 ml
with 1-naphthylphosphate.
234 TABLE
1
Determination of variousenzymes by the proposed methods Alkaline phosphatase (mU/7.5 ml”) 13.8 14.0
Given
Found
38.5 40.1
49.2 46.8
69.2 72.1
Sorbitol dehydrogenase (mU/7.5 1.40 1.35
Given Found
2.‘iS 2.78
0.52 0.62
Given Found
100.0 101.5
120.0 122.9
147.7 146.9
141.7 146.9
3.54 3.46
4.40 4.33
5.60 5.49
5.64 5.72
7.71 7.79
8.39 8.39
10.52 10.54
ml=)
1.02
1.56
2.08
2.60
3.12
3.64
4-16
4.68
5.20
1.09
1.51
2.11
2.55
3.10
3.70
4.20
4.68
5.10
Pseudocholinesterase (mU/8.0 Given Found
95-4 93.5
rnlb)
3.24 3.30
Horse-radish peroxidase (mU/7.5
73.8 73.5
mid)
9.8
19.6
29.4
39.2
49.0
58.8
68.6
78.4
88.2
107.8
8.8
18.6
29.6
40.2
49.0
58.8
68.4
76.4
87.2
108.0
=1 u = 40 p-nitrophenylphosphate (Boehringer). Cl U = 500 guaiacol
units (Boehringer). units (Boehringer).
bl U = 20 fructose/NADH dl U = 1000 butyrylcholine
units units
(Sigma).
Rr = 100
kohm)
is chosen
so as to maintain
a constant
concentration
of
4 PM NADH. The plot of tan u (ordinate) versus enzyme concentration is linear over the range specified and cuts the ordinate at tan Q! = 0.05. Table 1 gives some results for the determination of SDH_
Determination
of horse-radish peroxidase
with scopoletine
Horse-radish peroxidase catalyzes the oxidation of fluorescent scopoletine (6-methoxy-7-hydroxycoumarin) to form non-fluorescent oxidation products [11,12] (F, = 365 nm, F2 = 458 nm). Procedure_ To the quartz cuvette are added 2 ml of sodium acetate buffer solution
(pH 4.3,
0.2 M), 2 ml of aqueous
hydrogen
peroxide
solution
(0.5
mg ml-‘) and 0.2-2 ml of a solution of horse-radish peroxidase (EC 1.11.1.7, 2.6 mU ml-‘) for preparing a calibration graph or a suitable amount of the sample
solution.
After
dilution
with
water to 7.5
ml, thermostatting
for
5 min and adjustment to 0 mV, addition of the t&ant (0.01 mg of scopoletine per ml of water; SERVA, Heidelberg) is started. The working potential (60 mV; RZ = 10 kohm) is chosen so as to maintain a constant concentration of 1.3 PM scopoletine. In this case, the plot of tan OLversus enzyme concentration is sigmoidal (Fig. 3). Some results for the determination of horse-radish peroxidase are given in Table 1.
235
Fig. 3. Calibration graph for thedetetination
of horse-radish peroxidase with scopoletine.
Measurements via a fluorescence quenching substance Pseudocholinesterase can be determined with non-fluorescent l-naphthylacetate, fluorescent l-naphthol being formed 1131. It was shown that this fluorescence can be quenched by erythrosine, added stepwise from the automatic burette (F, = 365 run; F, = 458 nm). Procedure. To the quartz cuvette are added 1 ml of sodium phosphate buffer solution (pH 8.0, 0.4 M) and 0.1-l ml of a solution of butyrylcholine&erase (EC 3.1.1.8, 100 mU ml -* ; Sigma, Miinchen) for preparing a cahbration graph or a definite amount of the sample solution. After dilution with water to 7.5 ml, and thermostatting for 5 min, 0.5 ml of 1-naphthylacetate (1 mg ml-’ in methyl cellosolve; Serva) is added, the millivoltmeter is zeroed and addition of the titrant (0.6 mg of erythrosine per ml of water) is started. The working potential (40 mV; Rf = 134 kohm) is chosen so as to maintain a constant fluorescence corresponding to about 42 PM 1-naphthol, the original concentration of 1-naphthylacetate being 0.33 mM. The plot cf tan Q! (ordinate) versus enzyme concentration is linear and cuts the ordinate at tan (Y = 0.36. Some results for the determination of pseudocholinesterase are given in Table 1. In a similar way, lipase from porcine pancreas can be determined with fluorescein diacetate as a non-fluorescent substrate [14] and erythrosine as a quencher for the fluorescence of the resulting fluorescein in phosphate buffer solution of pH 7.5. The resulting “titration” curves, however, bend quickly, thus showing that the system is rapidly deactivated.
236 REFERENCES 1 2 3 4
S. Pantel and H. Weiss, Anal. Chim. Acta, 109 (1979) 351. B. Tan and J. K_ Grime, Anal. Lett., 12 B (1979) 1551. J_ K. Grime and K. R. Lockhart, Anal- Chim. Acta, 106 (1979) 251. G. G. Guilbault, Practical Fiourescence; Theory, Methods and Techniques, M. Dekker, New York, 1973, p_ 277. 5 Th. Fijrster, 2. Elektrochem., 53 (1949) 93. 6 H. Lando!t and R. BZirnstein, Numerical Data and Functional Relationships in Science and Technology, New Series, Vol. 3, Springer-Verlag, 1967, p. 296. ‘7 S. Pantel and H_ Weiss, Anal_ Chim. Acta, 74 (1975) 275. 8 D. W. Moss, Clin. Chim. Acta, 5 (1960) 283; Biochem. J., 76 (1960) 32P. 9 H. U. Bergmeyer, Methoden der enzymatischen Analyse, 3. Aufl., Verlag Chemie, Weinheim, 1974, Vol. 1, p_ 601. 10 E. L. Wehry (Ed.), Modem Fluorescence Spectroscopy, Vol_ II, Heyden, London, 1976, p_ 57. 11 W. A. Andreae, Nature, 175 (1955) 859. 12 H. Perschke and E. Broda, Nature, 190 (1961) 257. 13 G_ G. Guilbault and D. N. Kramer, Anal. Chem., 37 (1965) 14 G. G_ Guilbault and D. N. Kramer, Anal. Chem., 36 (1964)
1675.
409.
in The Netberiands
Short Communication
A FLUORISTATIC METEIOD FOR THE DETEiRMINATION OF ALKALINE PHOSPHATASE, SORBITOL DEHYDROGENASE, PEROXIDASE AND PSEUDOCHOLIJY-ESTERASE
SIE;GBE;RT PANTEL
Lehrstuht fiir Analytische Chemie. Freiburg i.Br_ (W. Germany) (Received 25th
Chemisches
Laboratorium
der Uniuersit%,
January 1981)
Instrumentation for fluoristatic procedures and several applications are described. The simple device is used to determine alkaline phosphatase (7-150 mlJ), sorbitol dehydrogenase (l-10 z&J) and peroxidase (0.5-5 mu) with fluorescent substrates and pseudocbolinest (10-100 mu) with a non-fluorescent substrate and erythrosine as fluorescence quencher_ The method is of spec*& interest for the determination of enzymes with low substrate concentrations.
Summary_
The applicability of ‘%&at” methods in analyticaI chemistry for the determination of catalysts, activators and inhibitors has been reported quite recently [I--3] _ The ma& advantage of these open-system methods lies in the fact that well-defined, low concentrations of substrate or product can be kept constant for a much longer time than in closed systems. This is of special interest in enzymatic analysis, as has been pointed out earlier 111. Fluorimetry can be up to several orders of magnitude more sensitive than absorption spectrophotometry [4] _ It seemed therefore to be of interest to study the use of fluorescence for regulating the addition of a suitable reagent in a stat method. With the fluorist&ic technique described here, it is possible to determine inorganic catalysts and enzymes which react with a fluorescent substrate to form non-fluorescent products. This is done by controhed addition of the fluorescent substrate solution at a speed such that the fluorescence remains constant during the reaction time observed. The added volume per time unit is a measure of the catalyst concentration, the activity of the enzyme being given in units analogous to international units (1 I.U. = catalytic activity which transforms 1 JHIIO!of substrate per minute under the reaction conditions used). The technique can also be used to follow reactions that produce fluorescent products from non-fluorescent substrates. In this case, the increasing fluorescence is quenched by controlled addition of a suitable substance [5,6] at a speed such that again the fluorescence remains constant during the measuring time. This mode of working is not that of a conventional stat method; the substrate or product concentration is not kept constant during the reaction time, but a physicat property of the product is 0003-2670/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
diminished to a preset level. This method has the advantage that nonfluorescent substrates can be used for the determination of enzymes, and the disadvantage that the addition curves for the quenching substance are not linear over a long period of time, because the quenching effect is not linear with the added volume of the quenching solution. Some examples are given below for the determination of enzymes by means of fluorescent substrate. An example of the determination of an enzyme with a non-fIuorescent substrate producing a fluorescent reaction product is also described. Instrumentaticm The apparatus used (Fig. 1) consists of the Combi-Tifmtor 3 D (Metrohm, Herisau, Switzerland) [‘7] and the fluorescence unit of the MPI-System (McKee-Pedersen, Danville, USA). Procedure with a fluorescent substrate. Into the quartz cuvette (18 mm inner diameter) of the measuring compartment M (Fig. 1) a~ pippetted 2 ml of buffer sclution and water up to 7.5 ml. The well-stirred solution is thermostatted (circulating thermostat) for 5 min to 25.0 + O.I”C and the millivoltmeter is set to zero with the aid of the mV some (TJ), Now a definite volume of the appropriate titrant, equivalent to the substrate concentration to be kept constant in the enzyme reaction, is added to the cuvette from the l-ml burette of the Microdosigraph. For this titrant concentration, the millivoltmeter is set to 60 mV (most sensitive switching region; preset working potential) by means of the variable feed-back resistor Rf. After the insfxument has been set thus, the enzyme activity can be determined as described below, Enzyme and substrate solutions were stored at 0°C in the dark. Unless otherwise stated, enzymes and reagents were obtained from Bee_bringer, Mannheim.
Fig. 1. Schematic representation of the measuring/regulating circuit. M, Sample/fiIter holder SIP-1017 with magnetic stirrer NIP-1024; OA, Operational amplifier MP-1006; U, millivolt source MF-1008; 50-2 V; Ph, photomultiplier MP-1021; L, mercury lamp Hanau St. 41/220 V (Quarzlarnpen GmbH-Hanau, FRG) with stabilized voltage supply; mV, mV-meter E510 (Metrohm); Imp, irnpulsornac E473 (Metrohm); Bur./R.ec, microdosimat (l-ml burette) with recorder attached [Mekohm); F,, F,, optical interference fitters.
233 Determination of alkaline phosphatase with I-naphthylphosphate Alkaline phosphatase catalyzes the hydrolysis of l-naphthylphosphate
to form 1-naphthol [ 8]_ Both the substrate and the Ijroduct are fluorescent, but under suitable conditions (F, = 313 nm, F2 = 365 nm; Fig. l), the substrate can be measured selectively. Procedure. To the quartz cuvette are added 2 ml of sodium hydroxideglpcine buffer solution (pH 9.8, 0.2 M) and 0.05-l ml of a solution of alkaline phosphatase (EC 3.1.3.1, 0.15 U ml-‘) for preparing a calibration graph or a definite amount of the sample solution within the specified range. After dilution with water to 7.5 ml, this solution is thermostatted for 5 min. The millivoltmeter is set to zero and the titrant addition is started; an aqueous solution of monosodium-1-naphthylphosphate monohydrate (0.25 mg ml-l) (EGA, Steinheim) is used. The working potential (60 mV; Rf = 9 kohm) is chosen so as to maintain a constant substrate concentration of 25 PM 1-naphthylphosphate in the solution. A typical recorder plot is given in Fig. 2. The calibration graph (tan Q versus enzyme concentration) is slightly and smoothly curved towards the tan Q axis, but passes through the origin of the coordinates. Table 1 gives some results for the determination of alkaline phosphatase. Determination of sorbitol dehydrogenase adenine dinucleotide (NADH)
with
reduced
p-nicotinamide
Sorbitol dehydrogenase (SDH) catalyzes the reduction of D-fructose with NADH [I, 9, lo], which is fluorescent (F, = 365 nm, F, = 458 nm). The resulting NAD+ does not fluoresce under these conditions. Procedure. To the quartz cuvette are added 1 ml of sodium phosphate buffer solution (pH 6.1, 0.3 M), 2 ml of D-fructose solution (10 mg ml-‘) and 0.1-l ml of a solution of SDH (EC 1.1.1.14, 10 mU ml-l) for preparing a calibration graph or an appropriate amount of the sample solution_ After dilution with water to 7.5 ml, this solution is thermostatted for 5 min, the millivoltmeter is set to zero and addition of the titrant (0.1 mg of p-NADHNa2 per ml of water) is started. The working potential (60 mV;
, 3 min.
Fig. 2. Recordergraphfor the determination of 90 mU of alkalinephosphatase in 7.5 ml
with 1-naphthylphosphate.
234 TABLE
1
Determination of variousenzymes by the proposed methods Alkaline phosphatase (mU/7.5 ml”) 13.8 14.0
Given
Found
38.5 40.1
49.2 46.8
69.2 72.1
Sorbitol dehydrogenase (mU/7.5 1.40 1.35
Given Found
2.‘iS 2.78
0.52 0.62
Given Found
100.0 101.5
120.0 122.9
147.7 146.9
141.7 146.9
3.54 3.46
4.40 4.33
5.60 5.49
5.64 5.72
7.71 7.79
8.39 8.39
10.52 10.54
ml=)
1.02
1.56
2.08
2.60
3.12
3.64
4-16
4.68
5.20
1.09
1.51
2.11
2.55
3.10
3.70
4.20
4.68
5.10
Pseudocholinesterase (mU/8.0 Given Found
95-4 93.5
rnlb)
3.24 3.30
Horse-radish peroxidase (mU/7.5
73.8 73.5
mid)
9.8
19.6
29.4
39.2
49.0
58.8
68.6
78.4
88.2
107.8
8.8
18.6
29.6
40.2
49.0
58.8
68.4
76.4
87.2
108.0
=1 u = 40 p-nitrophenylphosphate (Boehringer). Cl U = 500 guaiacol
units (Boehringer). units (Boehringer).
bl U = 20 fructose/NADH dl U = 1000 butyrylcholine
units units
(Sigma).
Rr = 100
kohm)
is chosen
so as to maintain
a constant
concentration
of
4 PM NADH. The plot of tan u (ordinate) versus enzyme concentration is linear over the range specified and cuts the ordinate at tan Q! = 0.05. Table 1 gives some results for the determination of SDH_
Determination
of horse-radish peroxidase
with scopoletine
Horse-radish peroxidase catalyzes the oxidation of fluorescent scopoletine (6-methoxy-7-hydroxycoumarin) to form non-fluorescent oxidation products [11,12] (F, = 365 nm, F2 = 458 nm). Procedure_ To the quartz cuvette are added 2 ml of sodium acetate buffer solution
(pH 4.3,
0.2 M), 2 ml of aqueous
hydrogen
peroxide
solution
(0.5
mg ml-‘) and 0.2-2 ml of a solution of horse-radish peroxidase (EC 1.11.1.7, 2.6 mU ml-‘) for preparing a calibration graph or a suitable amount of the sample
solution.
After
dilution
with
water to 7.5
ml, thermostatting
for
5 min and adjustment to 0 mV, addition of the t&ant (0.01 mg of scopoletine per ml of water; SERVA, Heidelberg) is started. The working potential (60 mV; RZ = 10 kohm) is chosen so as to maintain a constant concentration of 1.3 PM scopoletine. In this case, the plot of tan OLversus enzyme concentration is sigmoidal (Fig. 3). Some results for the determination of horse-radish peroxidase are given in Table 1.
235
Fig. 3. Calibration graph for thedetetination
of horse-radish peroxidase with scopoletine.
Measurements via a fluorescence quenching substance Pseudocholinesterase can be determined with non-fluorescent l-naphthylacetate, fluorescent l-naphthol being formed 1131. It was shown that this fluorescence can be quenched by erythrosine, added stepwise from the automatic burette (F, = 365 run; F, = 458 nm). Procedure. To the quartz cuvette are added 1 ml of sodium phosphate buffer solution (pH 8.0, 0.4 M) and 0.1-l ml of a solution of butyrylcholine&erase (EC 3.1.1.8, 100 mU ml -* ; Sigma, Miinchen) for preparing a cahbration graph or a definite amount of the sample solution. After dilution with water to 7.5 ml, and thermostatting for 5 min, 0.5 ml of 1-naphthylacetate (1 mg ml-’ in methyl cellosolve; Serva) is added, the millivoltmeter is zeroed and addition of the titrant (0.6 mg of erythrosine per ml of water) is started. The working potential (40 mV; Rf = 134 kohm) is chosen so as to maintain a constant fluorescence corresponding to about 42 PM 1-naphthol, the original concentration of 1-naphthylacetate being 0.33 mM. The plot cf tan Q! (ordinate) versus enzyme concentration is linear and cuts the ordinate at tan (Y = 0.36. Some results for the determination of pseudocholinesterase are given in Table 1. In a similar way, lipase from porcine pancreas can be determined with fluorescein diacetate as a non-fluorescent substrate [14] and erythrosine as a quencher for the fluorescence of the resulting fluorescein in phosphate buffer solution of pH 7.5. The resulting “titration” curves, however, bend quickly, thus showing that the system is rapidly deactivated.
236 REFERENCES 1 2 3 4
S. Pantel and H. Weiss, Anal. Chim. Acta, 109 (1979) 351. B. Tan and J. K_ Grime, Anal. Lett., 12 B (1979) 1551. J_ K. Grime and K. R. Lockhart, Anal- Chim. Acta, 106 (1979) 251. G. G. Guilbault, Practical Fiourescence; Theory, Methods and Techniques, M. Dekker, New York, 1973, p_ 277. 5 Th. Fijrster, 2. Elektrochem., 53 (1949) 93. 6 H. Lando!t and R. BZirnstein, Numerical Data and Functional Relationships in Science and Technology, New Series, Vol. 3, Springer-Verlag, 1967, p. 296. ‘7 S. Pantel and H_ Weiss, Anal_ Chim. Acta, 74 (1975) 275. 8 D. W. Moss, Clin. Chim. Acta, 5 (1960) 283; Biochem. J., 76 (1960) 32P. 9 H. U. Bergmeyer, Methoden der enzymatischen Analyse, 3. Aufl., Verlag Chemie, Weinheim, 1974, Vol. 1, p_ 601. 10 E. L. Wehry (Ed.), Modem Fluorescence Spectroscopy, Vol_ II, Heyden, London, 1976, p_ 57. 11 W. A. Andreae, Nature, 175 (1955) 859. 12 H. Perschke and E. Broda, Nature, 190 (1961) 257. 13 G_ G. Guilbault and D. N. Kramer, Anal. Chem., 37 (1965) 14 G. G_ Guilbault and D. N. Kramer, Anal. Chem., 36 (1964)
1675.
409.