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Assay of cholinesterase in an electrode system with an immobilized substrate
Analytica
Chimica
OElsevier Scientific
85 (1976) 295-300 Publishing Company, Amsterdam
Acta,
ASSAY OF CHOLINESTERASE IMMOBILIZED SUBSTRATE
- Printed in The Netherlands
IN AN ELECTRODE
SYSTEM
WITH AN
G. G. GUILBAULT and A. IWASE Department (U.S.A.)
(Received
of Chemistry,
19th February
University
of New
Orleans,
New
Orleans,
Louisiana
70122
1976)
SUMMARY A rapid, simple method for the measurement of cholincsterase based acetylcholine substrate is described. Each assay requires only 3 min and substrate can be used for 10 assays with excellent results; the substrate renewed easily and quickly. The precision obtained (2.5 ‘3%)is the same with the soluble substrate system.
on an immobilized the immobilized can then be as that po.ssible
Several methods of assay for cholinesterase based on detection of the increase in hydrogen ion during the enzymatic reaction have been reported [l-3] .Recently, a rapid method for the measurement of cholinestcrase was developed by Guilbault and Gibson [4], who were able to analyze 40 samples per hour in an automated system. This method was based on the observation that under certain conditions an enzymatic reaction can result in a linear change of pH with time. In this paper, the concept of the use of an immobilized substrate for assay of enzyme activity with a hydrogen-ion electrode is reported. It is easy to design a reaction cell for use with an immobilized substrate, and it was found possible to use an immobilized substrate system for electroanalytical measurement of the cholinestcrase by means of the following reaction: [CII,CqCH,CII2N+(CH,),],
+ II20 * CH,COO- + H+ + [ HOCH,CH,N+(CH,),
lH
where subscript R indicates the resin phase used to insolubilize (immobilize) the substrate. EXPERIMENTAL
Immobilization of substrate
Acetylcholine chloride (2 g; Calbiochem Co., Calif.) and a cation-exchange resin (1 g; Na’ form; Dowex 50, 200 mesh, or Amberlite IRC-50, 2-50
296
mesh) were added to about 0.5 ml of 2 mM Tris buffer solution (pH 8.10), and the slurry was mixed well. The mixture was filtered on a glass filter, and washed three times with the same buffer solution. This immobilized substrate was kept in a refrigerator until use. Reagents
Tris buffer, pH 8.1CP8.15, 2 mM, was prepared fresh daily from tris(hydroxymcthyl)aminomethane (Sigma, St. Louis). Sodium chloride was added to insure a constant ionic strength (cc) and to stabilize the electrode response. The cholinesterase enzyme (Worthington Biochemical Co., Freehold, N-J.; 5 U/mg) was dissolved in redistilled water (10 U/100 ~1). Apparatus A Coming
Digital 110 pH meter or a Beckman Research pH meter was combined with a Heath operational amplifier system. pH-time cures were recorded with a Heath recorder adjusted so that 0.1 pH unit corresponded to 6.5 cm on the paper. The two reaction cells (Fig. 1) were constructed from polyethylene beakers (diameter, 2 cm; height, 3 cm) with tightly fitting lids. Holes were made in the lid so that a flat-tipped glass electrode [ 51 (Radiometer E 5036/O; ca. 12 mm’ area) and glass tube A could be placed in it. An additional hole B was used for the addition of a sample with a micropipette. The immobilized substrate was placed between layers of nylon cloth (Nylon 677-74) and secured by rubber O-rings. For cell 1, 100 mg of immobilized substrate on Amberlite IRC50, and for cell 2, 1 g of immobilized substrate on Dowex 50, were used. Teflon-coated magnetic bars were used to stir all solutions. Procedures
To a reaction cell, was added 3.0 ml of Tris buffer. When a steady pH reading was observed, 100 ~1 of the enzyme sample was rapidly added and
bcr 2 Fig. 1. Reaction
cells used.
For description,
see text.
297
the pH change was recorded for ca. 2 min. All experiments were conducted at room temperature (23 “C). The immobilized substrate (reaction cell 2) could be used 10 times, and then an easy renewal of the substrate resin was effected. Some results were obtained in an atmosphere of nitrogen (see below). RESULTS
AND
DISCUSSION
The two different reaction cells are shown in Fig. 1. Cell 1 is a selfcontained “substrate” electrode, with the immobilized substrate directly on the surface of the glass electrode, kept in place by nylon net in a “basket”. Configuration 2 uses the immobilized enzyme held in a nylon net “basket” at the bottom of the cell, with the electrode then free to measure H* ions. Some typical pH-time curves obtained with those cells, and no cholinesterase added are shown in Fig. 2. No change in pH was observed with cell 2, but with cell 1, a increase in initial pH was observed, with a leveling after about 8-10 min. Some typical rate curves obtained with cells 1 and 2 upon addition of choline&erase are shown in Fig. 3. Curve 1 represents the rate curve upon addition of 0.5 units of cholinesterase obtained with the reaction cell 1 in 2 mM Tris buffer (pH 8.10, /.I= 0.01). Curves 2 and 3 show the rate curves after addition of 0.5 and 0.25 units of cholinesterase, and were obtained with reaction cell 2 in 2 mM Tris buffer. Under these conditions, the enzymatic reaction yields a linear change of pH with time. In the case of cell 2, it was necessary to wash with distilled water three times before each use.
Fig. 2. Spontaneous pH-time curves obtained in 3.0 ml of 2 mM Tris buffer pH 8.15, JJ= 0.01. I. Reaction Cell 1. II. Reaction Cell 2. Fig. 3. Rate curves obtained on addition of 100 ~1 of enzyme sample to 3.0 ml of 2 mM ‘I&s buffer (pH 8.10, M = 0.01). I. 0.50 units cholinesterase, Cell 1. II. 0.50 units cholinesterase, Cell 2. III. 0.25 units cholincsterase, Cell 2.
298
The effect of PH. ionic strength and buffer concentration Figure 4 shows the pH profile for the reaction rate of 0.5 units of cholinesterase, in the 2 mM Tris buffer solution, containing 0.01 M NaCl, the pH of which was adjusted from 7.5 to 8.5 by adding 0.1 M hydrochloric acid. It is clear that the reaction rate is constant over the range pH 7.8-8.3 in both cases. The rate cumes were measured in reaction cell 2 under the same conditions as for Fig. 4 at pH 8.18, except for the ionic strength used. An ideal reaction curve was not obtained in reaction cell 1, at varying concentrations of sodium chloride (O-02--0.06 M). Figure 5 shows the plot of the reaction rate against the square root of the ionic strength. An approximately straight line with a slope of -0.4 was obtained. The reaction rate decreased as the concentration of sodium chloride increased; this can be attributed to the decrease in activity of the substrate on the resin phase. The rate curves were then measured under the same conditions as Fig. 4 at pH 8.10, except for the use of varying concentrations of Tris buffer from 2 to 8 mM. In both cell 1 and cell 2, the region of linearity of the rate curves decreased as the concentration of Tris buffer increased. Consequently, the concentration of buffer must be limited, because of this lowering of sensitivity to hydrogen ion. Good results were obtained with 2 mM Tris buffer. The effect of enzyme concentmtions Figure 6 illustrates some calibration plots obtained_ The sensitivity of the immobilized substrate method with cell 1 was found to be higher than that of the cell 2. IJnfortunately, cell 1 is inefficient. For cell 2, the sensitivity was found to be the same as that of the soluble substrate method, and the
m
,..‘
I..
-.--.--.-..---1 0
;
F 1. : i ,
‘.:
“I
b
Fig. 4. Effect of pH on the rate of addition of 0.50 units of cholinesterase to
2 mM Tris buffer, ~1= 0.01. I. Cell 1. II. Cell 2.
3.0 ml of
Fig. 5. Effect of ionic strength on the rate in cell 2 at pH 8.15. Same conditions as Fig. 4.
299
method is easy to run. A linear plot was obtained in the enzyme concentration range 0.025-l-O units per 100 ~1 sample. In the range above 1.0 units, the reproducibility of the rate curves was not good, probably because the reaction rate was too fast. The rate curve with ccl1 2, even with the closed system (no nitrogen), was the same as that obtained in nitrogen. The relationship between the reaction rate obtained with a particular system and the number of runs is given in Fig. 7. As shown for cell 2, for a 0.5unit cholincsterase sample, the same reading was obtained for 10 assays, followed by a slight decrease up to 18 assays. At point R of curve 2, the resin was renewed by passing a 10 ‘% acetylcholine chloride solution through it; good results were then obtained, as is shown by Curve III of Fig. 7. However, after another 10 assays, another regeneration was necessary. Only a few minutes are required to renew the resin. Hence, this method is useful as a practical assay device, although it does have one weak point, in that the kinetic data are affected quite sensitively by variations in the ionic strength. Conclusions Compared to the mobile substrate system described previously [ 41 for cholinesterase assay, this type of approach with cell 2 has some advantages: it is fast (3 min per assay) and easy to perform. An immobilized substnte can be prepared easily from acctylcholine chloride and the sodium form of a cation resin, and used for a number of repetitive assays. Cell 1 suffers from a continuous loss of substrate after 2-3 runs, and hence with this configuration the substrate layer must be renewed aftm each run. The financial assistance of the General Medicine Division, National Institute of Health, Grant No. GM 17268, is gratefully acknowledged_
‘2,
Fig. 6. Calibration II. Cell 2. oResuIts
‘CC
ml
,-IT3.2 c
curves fur cholinesterase. 2 mM Tris buffer, obtained without nitrogen.
p1I 8.10,
p = 0.01.
I. Cell
1.
Fig. 7. Stability of immobilized substrate preparations. 2 mNI his buffer, pII 8.10 ? 0.05. p = 0.01. 0.5 U/ZOO ~1 cholinesterase added. I. Cell 1. II. Cell 2. III. Cell 2, after renewal of substrate layer. without nitrogen.
REFERENCES 1 Ii. 0. Michel, J. Lab. Clin. Med., 34 (1949) 1566. 2 J. Chouteau, P. Rancien and A. Karamanicin, Bull. Sot.
Chim. Biol., 38 (1956) 3 K. I,. Crochet and J. G. Montalvo, Anal. Chim. Acta. 66 (1973) 259. 4 K. Gibson and G. G. Guilbault. Anal. Chim. Acta, 76 (1975) 245. 5 G. G. Guilbault and M. Tarp, Anal. Chim. Acta. 73 (1974) 355.
139.
Chimica
OElsevier Scientific
85 (1976) 295-300 Publishing Company, Amsterdam
Acta,
ASSAY OF CHOLINESTERASE IMMOBILIZED SUBSTRATE
- Printed in The Netherlands
IN AN ELECTRODE
SYSTEM
WITH AN
G. G. GUILBAULT and A. IWASE Department (U.S.A.)
(Received
of Chemistry,
19th February
University
of New
Orleans,
New
Orleans,
Louisiana
70122
1976)
SUMMARY A rapid, simple method for the measurement of cholincsterase based acetylcholine substrate is described. Each assay requires only 3 min and substrate can be used for 10 assays with excellent results; the substrate renewed easily and quickly. The precision obtained (2.5 ‘3%)is the same with the soluble substrate system.
on an immobilized the immobilized can then be as that po.ssible
Several methods of assay for cholinesterase based on detection of the increase in hydrogen ion during the enzymatic reaction have been reported [l-3] .Recently, a rapid method for the measurement of cholinestcrase was developed by Guilbault and Gibson [4], who were able to analyze 40 samples per hour in an automated system. This method was based on the observation that under certain conditions an enzymatic reaction can result in a linear change of pH with time. In this paper, the concept of the use of an immobilized substrate for assay of enzyme activity with a hydrogen-ion electrode is reported. It is easy to design a reaction cell for use with an immobilized substrate, and it was found possible to use an immobilized substrate system for electroanalytical measurement of the cholinestcrase by means of the following reaction: [CII,CqCH,CII2N+(CH,),],
+ II20 * CH,COO- + H+ + [ HOCH,CH,N+(CH,),
lH
where subscript R indicates the resin phase used to insolubilize (immobilize) the substrate. EXPERIMENTAL
Immobilization of substrate
Acetylcholine chloride (2 g; Calbiochem Co., Calif.) and a cation-exchange resin (1 g; Na’ form; Dowex 50, 200 mesh, or Amberlite IRC-50, 2-50
296
mesh) were added to about 0.5 ml of 2 mM Tris buffer solution (pH 8.10), and the slurry was mixed well. The mixture was filtered on a glass filter, and washed three times with the same buffer solution. This immobilized substrate was kept in a refrigerator until use. Reagents
Tris buffer, pH 8.1CP8.15, 2 mM, was prepared fresh daily from tris(hydroxymcthyl)aminomethane (Sigma, St. Louis). Sodium chloride was added to insure a constant ionic strength (cc) and to stabilize the electrode response. The cholinesterase enzyme (Worthington Biochemical Co., Freehold, N-J.; 5 U/mg) was dissolved in redistilled water (10 U/100 ~1). Apparatus A Coming
Digital 110 pH meter or a Beckman Research pH meter was combined with a Heath operational amplifier system. pH-time cures were recorded with a Heath recorder adjusted so that 0.1 pH unit corresponded to 6.5 cm on the paper. The two reaction cells (Fig. 1) were constructed from polyethylene beakers (diameter, 2 cm; height, 3 cm) with tightly fitting lids. Holes were made in the lid so that a flat-tipped glass electrode [ 51 (Radiometer E 5036/O; ca. 12 mm’ area) and glass tube A could be placed in it. An additional hole B was used for the addition of a sample with a micropipette. The immobilized substrate was placed between layers of nylon cloth (Nylon 677-74) and secured by rubber O-rings. For cell 1, 100 mg of immobilized substrate on Amberlite IRC50, and for cell 2, 1 g of immobilized substrate on Dowex 50, were used. Teflon-coated magnetic bars were used to stir all solutions. Procedures
To a reaction cell, was added 3.0 ml of Tris buffer. When a steady pH reading was observed, 100 ~1 of the enzyme sample was rapidly added and
bcr 2 Fig. 1. Reaction
cells used.
For description,
see text.
297
the pH change was recorded for ca. 2 min. All experiments were conducted at room temperature (23 “C). The immobilized substrate (reaction cell 2) could be used 10 times, and then an easy renewal of the substrate resin was effected. Some results were obtained in an atmosphere of nitrogen (see below). RESULTS
AND
DISCUSSION
The two different reaction cells are shown in Fig. 1. Cell 1 is a selfcontained “substrate” electrode, with the immobilized substrate directly on the surface of the glass electrode, kept in place by nylon net in a “basket”. Configuration 2 uses the immobilized enzyme held in a nylon net “basket” at the bottom of the cell, with the electrode then free to measure H* ions. Some typical pH-time curves obtained with those cells, and no cholinesterase added are shown in Fig. 2. No change in pH was observed with cell 2, but with cell 1, a increase in initial pH was observed, with a leveling after about 8-10 min. Some typical rate curves obtained with cells 1 and 2 upon addition of choline&erase are shown in Fig. 3. Curve 1 represents the rate curve upon addition of 0.5 units of cholinesterase obtained with the reaction cell 1 in 2 mM Tris buffer (pH 8.10, /.I= 0.01). Curves 2 and 3 show the rate curves after addition of 0.5 and 0.25 units of cholinesterase, and were obtained with reaction cell 2 in 2 mM Tris buffer. Under these conditions, the enzymatic reaction yields a linear change of pH with time. In the case of cell 2, it was necessary to wash with distilled water three times before each use.
Fig. 2. Spontaneous pH-time curves obtained in 3.0 ml of 2 mM Tris buffer pH 8.15, JJ= 0.01. I. Reaction Cell 1. II. Reaction Cell 2. Fig. 3. Rate curves obtained on addition of 100 ~1 of enzyme sample to 3.0 ml of 2 mM ‘I&s buffer (pH 8.10, M = 0.01). I. 0.50 units cholinesterase, Cell 1. II. 0.50 units cholinesterase, Cell 2. III. 0.25 units cholincsterase, Cell 2.
298
The effect of PH. ionic strength and buffer concentration Figure 4 shows the pH profile for the reaction rate of 0.5 units of cholinesterase, in the 2 mM Tris buffer solution, containing 0.01 M NaCl, the pH of which was adjusted from 7.5 to 8.5 by adding 0.1 M hydrochloric acid. It is clear that the reaction rate is constant over the range pH 7.8-8.3 in both cases. The rate cumes were measured in reaction cell 2 under the same conditions as for Fig. 4 at pH 8.18, except for the ionic strength used. An ideal reaction curve was not obtained in reaction cell 1, at varying concentrations of sodium chloride (O-02--0.06 M). Figure 5 shows the plot of the reaction rate against the square root of the ionic strength. An approximately straight line with a slope of -0.4 was obtained. The reaction rate decreased as the concentration of sodium chloride increased; this can be attributed to the decrease in activity of the substrate on the resin phase. The rate curves were then measured under the same conditions as Fig. 4 at pH 8.10, except for the use of varying concentrations of Tris buffer from 2 to 8 mM. In both cell 1 and cell 2, the region of linearity of the rate curves decreased as the concentration of Tris buffer increased. Consequently, the concentration of buffer must be limited, because of this lowering of sensitivity to hydrogen ion. Good results were obtained with 2 mM Tris buffer. The effect of enzyme concentmtions Figure 6 illustrates some calibration plots obtained_ The sensitivity of the immobilized substrate method with cell 1 was found to be higher than that of the cell 2. IJnfortunately, cell 1 is inefficient. For cell 2, the sensitivity was found to be the same as that of the soluble substrate method, and the
m
,..‘
I..
-.--.--.-..---1 0
;
F 1. : i ,
‘.:
“I
b
Fig. 4. Effect of pH on the rate of addition of 0.50 units of cholinesterase to
2 mM Tris buffer, ~1= 0.01. I. Cell 1. II. Cell 2.
3.0 ml of
Fig. 5. Effect of ionic strength on the rate in cell 2 at pH 8.15. Same conditions as Fig. 4.
299
method is easy to run. A linear plot was obtained in the enzyme concentration range 0.025-l-O units per 100 ~1 sample. In the range above 1.0 units, the reproducibility of the rate curves was not good, probably because the reaction rate was too fast. The rate curve with ccl1 2, even with the closed system (no nitrogen), was the same as that obtained in nitrogen. The relationship between the reaction rate obtained with a particular system and the number of runs is given in Fig. 7. As shown for cell 2, for a 0.5unit cholincsterase sample, the same reading was obtained for 10 assays, followed by a slight decrease up to 18 assays. At point R of curve 2, the resin was renewed by passing a 10 ‘% acetylcholine chloride solution through it; good results were then obtained, as is shown by Curve III of Fig. 7. However, after another 10 assays, another regeneration was necessary. Only a few minutes are required to renew the resin. Hence, this method is useful as a practical assay device, although it does have one weak point, in that the kinetic data are affected quite sensitively by variations in the ionic strength. Conclusions Compared to the mobile substrate system described previously [ 41 for cholinesterase assay, this type of approach with cell 2 has some advantages: it is fast (3 min per assay) and easy to perform. An immobilized substnte can be prepared easily from acctylcholine chloride and the sodium form of a cation resin, and used for a number of repetitive assays. Cell 1 suffers from a continuous loss of substrate after 2-3 runs, and hence with this configuration the substrate layer must be renewed aftm each run. The financial assistance of the General Medicine Division, National Institute of Health, Grant No. GM 17268, is gratefully acknowledged_
‘2,
Fig. 6. Calibration II. Cell 2. oResuIts
‘CC
ml
,-IT3.2 c
curves fur cholinesterase. 2 mM Tris buffer, obtained without nitrogen.
p1I 8.10,
p = 0.01.
I. Cell
1.
Fig. 7. Stability of immobilized substrate preparations. 2 mNI his buffer, pII 8.10 ? 0.05. p = 0.01. 0.5 U/ZOO ~1 cholinesterase added. I. Cell 1. II. Cell 2. III. Cell 2, after renewal of substrate layer. without nitrogen.
REFERENCES 1 Ii. 0. Michel, J. Lab. Clin. Med., 34 (1949) 1566. 2 J. Chouteau, P. Rancien and A. Karamanicin, Bull. Sot.
Chim. Biol., 38 (1956) 3 K. I,. Crochet and J. G. Montalvo, Anal. Chim. Acta. 66 (1973) 259. 4 K. Gibson and G. G. Guilbault. Anal. Chim. Acta, 76 (1975) 245. 5 G. G. Guilbault and M. Tarp, Anal. Chim. Acta. 73 (1974) 355.
139.