- Email: [email protected]
A colorimetric micro method for the determination of cholinesterase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 7% 1-7 (1959)
A Calorimetric Micro Method for the Determination of Cholinesterase’ Don E. McOsker’ and Louise J. Daniel From the Department of Biochemistry and Nutrition, Cornell University, Ithaca, New York
Received June 16, 1958
Of the many methods that have been described for the determination of cholinesterases, most have employed the various “normal” esters of choline. The possibility of using the thioesters of choline, which are hydrolyzed to acetic acid and thiocholine by cholinesterases, was suggested by the histochemical work of Koelle (I). The present method utilizes acetylthiocholine as the substrate. The determination of the resultant sulfhydryl groups by sodium nitroprusside constitutes a direct measure of the enzyme activity. As this work was nearing completion, Augustinsson (2) published a method using thioesters as substrates. He determined the sulfhydryl groups formed by an iodometric titration. Meyer and Wilbrandt (3) have also used thiocholine esters and the iodometric titration to determine cholinesterase in blood and plasma. Following the initial report of this method,l Tabachnick (4) and Gal and Roth (5) reported methods utilizing esters of thiocholine in which the enzymic reaction was followed by the decrease in absorbance of the thioester bond. The method to be presented allows the determination of cholinesterase activity with high precision in a minimum amount of time. It is a highly sensitive method and the reaction is followed by measuring the appearancr of product and not the disappearance of substrate. METHOD The following reagents are pipetted into a 15.ml. centrifuge tube: 0.20 ml. of iodide 3; 0.50 ml. of 0.125 M tris(hydroxymethyl)amino0.015 M scetylthiocholine methane (Tris) buffer, pH 7.4; 0.20 ml. of 3.5 M sodium chloride, and water to a -. r A portion of these data were presented at the 47th meeting of the American Society of Biological Chemists, at Atlantic City, N. J., April 1956. An abstract appeared in Federation Proc. 16, 313 (1956). * Present address: Procter and Gamble Company, Cincinnati, Ohio. 3 Obtained from Mann Fine Chemicals Company, New York, N. Y.
2
MCOSKER
AND DANIEL
tot,al volume of 1.30 ml. After equilibrating to constant temperature (37”C.j, 0.10 ml. of a tissue homogenate is added. At the end of 10 min. incubation, 0.10 ml. of 25% (w/v) trichloroacetic acid (TCA) is added to stop the reaction and to precipitate the protein. (If whole blood is used as the enzyme source, it is usually necessary to add 0.10 ml. of 50% (w/v) TCA to obtain a clear supernatant. This amount of TCA does not interfere with the subsequent sulfhydryl det,ermination.) The reaction mixture is next centrifuged in a clinical centrifuge for about 5 min. An aliquot of the clear supernatant is withdrawn, and the sulfhydryl (SH) content is determined using the modification* of the method of Grunert and Phillips (6)) as described below. The following reagents are pipetted into 3.0.ml. Beckman cuvettes: 2.0 ml. saturated NaCl; 0.4 ml. of Na&03-NaCN solution containing 21.2 g. anhydrous NaLXL and 0.44 g. NaCN/lOO ml.; and 0.4 ml. sodium nitroprusside solution containing 27 mg./ml. The reagents are mixed, and 0.2 ml. of sample is added immediately. Appropriate blanks are made by omitting either the enzyme or the substrate from the incubation mixture. The optical density is measured exactly 30 sec. after t,he sample is added in the Beckman spectrophotometer, model DIJ, at, a wavelength of 520 rnp. RESULTS
AND
Discussion-
Koelle (l), using histochemical techniques, reported that the cholinesterases show the same pattern of specificity toward the thiocholine esters as for the nonthioesters of choline. However, since these techniques are difficult to quantitate, it seemed advisable to study various properties of the enzyme reaction using the calorimetric procedure and acetylt,hiocholine as the substrate. Enzyme activity is plotted against enzyme concentration in Fig. 1. The linear plot shows there was strict proportionulit’y between the enzyme concentration and enzyme activity. Figure 2 shows the linearity of the enzyme reaction with respect to time. It can be seen that the reaction was linear for 15 min. with a grad& decrease in rate thereafter. The reaction reached equilibrium at about 45 min. After 30 min. of incubation, 45% of the substrate had been hydrolyzed, and at the end of 45 min. 50% of the substrate was split. Any incubation time up to 15 min. would yield precise measurement of enzyme activity. The optimum substrate concentration is shown in Fig. 3. The maximum activity was observed at approximately 2 X 1O-3 M acetylthiocholine in the absence of added sodium chloride. This concentration is somewhat lower than most investigators report for acetylcholine when the activity is measured using the Warburg apparatus, but it is in good agreement with the data of Smallman and Wolfe (7). These workers, using a purified enzyme preparation, determined the activity by titrating the acetic acid produced. They reported an optimum subst,rate concentration of 1 X 1Om-3M uoet,ylcholine in the presence of 0.05 dl sodium chloride. Using the colorimet,ric 4 R. W. Von Korff,
unpublished.
DETERMINATIOX
OF
3
CHOLINESTERASE
eo-
ml. ENZYME
FIG. 1. Relationship between enzyme activity and enzyme conrentration. A 10% rat, brain homogenate was the source of enzyme. .40.35.30.25-
5
IO
15
20
25
30
35
40
45
50
55
60
1 FIG. 2. Effect of time of incubat,ion on the sulfhydryl groups produced from 3.0 cmoles acetylthiocholine, as measured by change in optical density (A, ). -4 IWh rat brain homogenat’e was the source of enzyme. Time (Minutes
procedure, increasing the sodium chloride concentration up to 0.5 M increased the substrate optimum only slightly when homogenates were used as the source of enzyme. There is, however, a marked difference in the shape of the curves when acetylthiocholine and acetylcholine are compared as substrates. There is a sharp drop in activity as the acetylcholine concentration is increased beyond the optimum. This does not occur with acetylthiocholine. The possibility that the substrate was hydrolyzed by enzymes other than cholinesterases was eliminated by using eserine, a known inhibitor of cholin-
4
MCOSKER
AND
Molar Acetylthiochotiie
DANIEL
Iodide Cont. x IO’
FIG. 3. Effect of substrate concentration on enzyme activity; 0.30 ml. of a 10% rat brain homogenate was the source of enzyme.
282420-
FIQ.
4. Competitive
inhibition
by eserine. 0-O
no eserine; A---A
1 X l(re
M eserine; 0.30 ml. of a 10% rat brain homogenate was the source of enzyme. Activity was measured as micromoles SH liberated expressed as molarities.
in 10 min. Substrate concentrations
are
esterase. These data are presented in the form of a Lineweaver-Burk plot in Fig. 4. It is obvious from these results that eserine inhibited the hydrolysis of acetylthiocholine in a competitive manner. Inhibition was exhibited at concentrations down to 1 X 10-S M eserine; 6 X lo+ M eserine completely inhibited the reaction in brain homogenates at the substrate concentrations used.
DETERMINATION
OF
TABLE E#ect of Sodium
Chloride
I
on the Activation
of Cholinesterase
Enzyme activitya
10% rat brain homogenate
-N&l ad/10
ml.
0.05 0.20 0.40
5
CHOLINESTERASE
+NaCl
Increase in activity
0.117 0.378 0.603
42.7 38.5 24.8
min.
0.082 0.273 0.483
R
a Substrate concentration was 2 X 10e3M; NaCl, 4.66 X 10-l M. b Change in optical density. TABLE Amount
of Enzyme
Amount of fresh tissue”
Required
for
II
Calorimetric
Volume of incubation mixture
Determination
Substrate hydrolyzed/l0 min.
of Cholinesterase co, e..i&ent*/
fw.
ml.
Lmde
d.
24 8
1.5 0.5
0.375 0.125
8.4 2.8
n Chick embryo brain. b Calculated from micromoles of substrate hydrolyzed.
The effect of added sodium chloride on the activity of choline&erase is shown in Table I. It is apparent that sodium chloride caused considerable activation, particularly when the amount of homogenate was very small. The decrease in activation, which was noted as the quantity of homogenate was increased, is attributed to the endogenous salts present in the homogenate. No attempt was made to determine whether this activation was due to the added sodium ions or merely to the increase in the ionic strength*of the medium. The micro qualities of this method are best illustrated in Table 11’. If the final volume is reduced to 0.5 ml., quantities of enzyme that will hydrolyze 0.125 bmole substrate have been accurately measured. In the case of the chick embryo brain, 8 mg. fresh tissue was sufficient. Whole blood and blood plasma are tissues that contain considerable quantities of cholinesterase and are tissues in which cholinesterase is determined clinically. The results obtained for whole blood and blood plasma of dogs are presented in Table III. Agreement between duplicate samples was quite satisfactory. The differences among the dogs have no significance, since these were mongrel dogs for which no prior history was available. The calorimetric method was also used to determine the cholinesterase activity of the brain of developing chick embryos. These data are presented in Table IV. The results indicate that the level of cholinesterase in the
MCOSKER
AND
TABLE Choline&erase
Activity
DANIEL
III
of Whole Blood and Blood Plasma
of Dogs
Two-tenths milliliter of heparinized whole blood or plasma was used for each assay, and duplicate determinations (a and 5) were made. Cholinesterase activity Sample No.
noles SH/lO
Whole blood 1 2 3 4 5 Blood plasma 1 2 3 5
l-
Average
b
Average
/umoles
min
SE/hr./ml.
0.130 0.125 0.126 0.110 0.135
0.130 0.130 0.123 0.111 0.138
0.130 0.128 0.125 0.111 0.137
28.80 28.13 24.98 30.83
0.143 0.145 0.153 0.191
0.155 0.155 0.153 0.199
0.149 0.150 0.153 0.195
33.53 33.75 34.43 43.88
-I
-
emymc
29.25
-
TABLE
IV Cholinesterase Activity of the Brains of Developing Chick Embryos Each sample was a composite of five brains. The tissue was removed immediately after the embryo was sacrificed by decapitation and placed in 15 ml. of ice-cold distilled water. The pooled tissue was homogenized and 3 ml. withdrawn for assay. The values represent the average of duplicates which were consistently in excellent agreement. Sample No.
Age of embryos
dL%YS
1 2 3 4 5 6 7 8 9 10 11 12
12 12 12 15 15 15 18 18 18 21 21 21
Dry weight of five brains
g. 0.12 0.12 0.11 0.25 0.26 0.25 0.44 0.46 0.45 0.54 0.53 0.50
Cholinesterase activity per g. dry tissue pm&s
161 149 181 182 173 158 219 185 191 122 241 240
per five brains SH/lO
min.
19 18 20 46 45 40 97 85 86 66 128 120
DETERMINATION
OF
i
CHOLINESTERASE
embryonic chick brain increased consistently from the 12th through the 21st day of incubation. These observations are in accord with the data of Nachmansohn (8). Agreement among the three samples within a given age group was good, except for sample 10. No plausible explanation for this low value is available. ACKNOWLEDGMENTS
The authors are most grateful to Dr. L. C. Norris for supplying the chick embryos used in this work.
and the Poultry
Department,
SUMMARY
1. *4 calorimetric micro method for the determination of cholinesterase activity using acetylthiocholine as substrate has been developed. 2. The method is rapid, requires simple laboratory equipment, and is sensitive to small amounts of enzyme; the reaction is followed by measuring the appearance of one of the reaction products (thiocholine). 3. The method has been applied to the determination of the cholinesterase activity in rat brain, in whole blood and blood plasma of dogs, and in the brains of 12-, 15-, I%, and 21-day-old chick embryos.
1. HOELLE, CT. B., J. Pharmacol. Exptl. 7’herap. 100, 158 (1950). 2. ACJGUSTINSSON, K. B., Sand. J. Clin. & Lab. Invest. ‘I, 284 (1955). 3. MEYER, A., AND WILBRANDT, W., Helv. Physiol. et Pharmacol. Acta 12, 206 (1954). 4. TABACHNICK, I. I. A., Biochiflz. et Biophys. Acta 21, 580 (1956). 5. GAL, E. M., AND ROTH, E., C/in. Chim. Acta ‘2, 316 (1957). 6. GRUNERT, R. R., AND PHILLIPS, P. H., Arch. Biochem. 30, 217 (1951). 7. SMALLMAN, B. Pu'.,AND WOLFE, L. S.,Enzymologia1’7,133 (1954). 8. NACHMANSOHN,D., Bull. sot. chim. biol. 21, 761 (1939).
A Calorimetric Micro Method for the Determination of Cholinesterase’ Don E. McOsker’ and Louise J. Daniel From the Department of Biochemistry and Nutrition, Cornell University, Ithaca, New York
Received June 16, 1958
Of the many methods that have been described for the determination of cholinesterases, most have employed the various “normal” esters of choline. The possibility of using the thioesters of choline, which are hydrolyzed to acetic acid and thiocholine by cholinesterases, was suggested by the histochemical work of Koelle (I). The present method utilizes acetylthiocholine as the substrate. The determination of the resultant sulfhydryl groups by sodium nitroprusside constitutes a direct measure of the enzyme activity. As this work was nearing completion, Augustinsson (2) published a method using thioesters as substrates. He determined the sulfhydryl groups formed by an iodometric titration. Meyer and Wilbrandt (3) have also used thiocholine esters and the iodometric titration to determine cholinesterase in blood and plasma. Following the initial report of this method,l Tabachnick (4) and Gal and Roth (5) reported methods utilizing esters of thiocholine in which the enzymic reaction was followed by the decrease in absorbance of the thioester bond. The method to be presented allows the determination of cholinesterase activity with high precision in a minimum amount of time. It is a highly sensitive method and the reaction is followed by measuring the appearancr of product and not the disappearance of substrate. METHOD The following reagents are pipetted into a 15.ml. centrifuge tube: 0.20 ml. of iodide 3; 0.50 ml. of 0.125 M tris(hydroxymethyl)amino0.015 M scetylthiocholine methane (Tris) buffer, pH 7.4; 0.20 ml. of 3.5 M sodium chloride, and water to a -. r A portion of these data were presented at the 47th meeting of the American Society of Biological Chemists, at Atlantic City, N. J., April 1956. An abstract appeared in Federation Proc. 16, 313 (1956). * Present address: Procter and Gamble Company, Cincinnati, Ohio. 3 Obtained from Mann Fine Chemicals Company, New York, N. Y.
2
MCOSKER
AND DANIEL
tot,al volume of 1.30 ml. After equilibrating to constant temperature (37”C.j, 0.10 ml. of a tissue homogenate is added. At the end of 10 min. incubation, 0.10 ml. of 25% (w/v) trichloroacetic acid (TCA) is added to stop the reaction and to precipitate the protein. (If whole blood is used as the enzyme source, it is usually necessary to add 0.10 ml. of 50% (w/v) TCA to obtain a clear supernatant. This amount of TCA does not interfere with the subsequent sulfhydryl det,ermination.) The reaction mixture is next centrifuged in a clinical centrifuge for about 5 min. An aliquot of the clear supernatant is withdrawn, and the sulfhydryl (SH) content is determined using the modification* of the method of Grunert and Phillips (6)) as described below. The following reagents are pipetted into 3.0.ml. Beckman cuvettes: 2.0 ml. saturated NaCl; 0.4 ml. of Na&03-NaCN solution containing 21.2 g. anhydrous NaLXL and 0.44 g. NaCN/lOO ml.; and 0.4 ml. sodium nitroprusside solution containing 27 mg./ml. The reagents are mixed, and 0.2 ml. of sample is added immediately. Appropriate blanks are made by omitting either the enzyme or the substrate from the incubation mixture. The optical density is measured exactly 30 sec. after t,he sample is added in the Beckman spectrophotometer, model DIJ, at, a wavelength of 520 rnp. RESULTS
AND
Discussion-
Koelle (l), using histochemical techniques, reported that the cholinesterases show the same pattern of specificity toward the thiocholine esters as for the nonthioesters of choline. However, since these techniques are difficult to quantitate, it seemed advisable to study various properties of the enzyme reaction using the calorimetric procedure and acetylt,hiocholine as the substrate. Enzyme activity is plotted against enzyme concentration in Fig. 1. The linear plot shows there was strict proportionulit’y between the enzyme concentration and enzyme activity. Figure 2 shows the linearity of the enzyme reaction with respect to time. It can be seen that the reaction was linear for 15 min. with a grad& decrease in rate thereafter. The reaction reached equilibrium at about 45 min. After 30 min. of incubation, 45% of the substrate had been hydrolyzed, and at the end of 45 min. 50% of the substrate was split. Any incubation time up to 15 min. would yield precise measurement of enzyme activity. The optimum substrate concentration is shown in Fig. 3. The maximum activity was observed at approximately 2 X 1O-3 M acetylthiocholine in the absence of added sodium chloride. This concentration is somewhat lower than most investigators report for acetylcholine when the activity is measured using the Warburg apparatus, but it is in good agreement with the data of Smallman and Wolfe (7). These workers, using a purified enzyme preparation, determined the activity by titrating the acetic acid produced. They reported an optimum subst,rate concentration of 1 X 1Om-3M uoet,ylcholine in the presence of 0.05 dl sodium chloride. Using the colorimet,ric 4 R. W. Von Korff,
unpublished.
DETERMINATIOX
OF
3
CHOLINESTERASE
eo-
ml. ENZYME
FIG. 1. Relationship between enzyme activity and enzyme conrentration. A 10% rat, brain homogenate was the source of enzyme. .40.35.30.25-
5
IO
15
20
25
30
35
40
45
50
55
60
1 FIG. 2. Effect of time of incubat,ion on the sulfhydryl groups produced from 3.0 cmoles acetylthiocholine, as measured by change in optical density (A, ). -4 IWh rat brain homogenat’e was the source of enzyme. Time (Minutes
procedure, increasing the sodium chloride concentration up to 0.5 M increased the substrate optimum only slightly when homogenates were used as the source of enzyme. There is, however, a marked difference in the shape of the curves when acetylthiocholine and acetylcholine are compared as substrates. There is a sharp drop in activity as the acetylcholine concentration is increased beyond the optimum. This does not occur with acetylthiocholine. The possibility that the substrate was hydrolyzed by enzymes other than cholinesterases was eliminated by using eserine, a known inhibitor of cholin-
4
MCOSKER
AND
Molar Acetylthiochotiie
DANIEL
Iodide Cont. x IO’
FIG. 3. Effect of substrate concentration on enzyme activity; 0.30 ml. of a 10% rat brain homogenate was the source of enzyme.
282420-
FIQ.
4. Competitive
inhibition
by eserine. 0-O
no eserine; A---A
1 X l(re
M eserine; 0.30 ml. of a 10% rat brain homogenate was the source of enzyme. Activity was measured as micromoles SH liberated expressed as molarities.
in 10 min. Substrate concentrations
are
esterase. These data are presented in the form of a Lineweaver-Burk plot in Fig. 4. It is obvious from these results that eserine inhibited the hydrolysis of acetylthiocholine in a competitive manner. Inhibition was exhibited at concentrations down to 1 X 10-S M eserine; 6 X lo+ M eserine completely inhibited the reaction in brain homogenates at the substrate concentrations used.
DETERMINATION
OF
TABLE E#ect of Sodium
Chloride
I
on the Activation
of Cholinesterase
Enzyme activitya
10% rat brain homogenate
-N&l ad/10
ml.
0.05 0.20 0.40
5
CHOLINESTERASE
+NaCl
Increase in activity
0.117 0.378 0.603
42.7 38.5 24.8
min.
0.082 0.273 0.483
R
a Substrate concentration was 2 X 10e3M; NaCl, 4.66 X 10-l M. b Change in optical density. TABLE Amount
of Enzyme
Amount of fresh tissue”
Required
for
II
Calorimetric
Volume of incubation mixture
Determination
Substrate hydrolyzed/l0 min.
of Cholinesterase co, e..i&ent*/
fw.
ml.
Lmde
d.
24 8
1.5 0.5
0.375 0.125
8.4 2.8
n Chick embryo brain. b Calculated from micromoles of substrate hydrolyzed.
The effect of added sodium chloride on the activity of choline&erase is shown in Table I. It is apparent that sodium chloride caused considerable activation, particularly when the amount of homogenate was very small. The decrease in activation, which was noted as the quantity of homogenate was increased, is attributed to the endogenous salts present in the homogenate. No attempt was made to determine whether this activation was due to the added sodium ions or merely to the increase in the ionic strength*of the medium. The micro qualities of this method are best illustrated in Table 11’. If the final volume is reduced to 0.5 ml., quantities of enzyme that will hydrolyze 0.125 bmole substrate have been accurately measured. In the case of the chick embryo brain, 8 mg. fresh tissue was sufficient. Whole blood and blood plasma are tissues that contain considerable quantities of cholinesterase and are tissues in which cholinesterase is determined clinically. The results obtained for whole blood and blood plasma of dogs are presented in Table III. Agreement between duplicate samples was quite satisfactory. The differences among the dogs have no significance, since these were mongrel dogs for which no prior history was available. The calorimetric method was also used to determine the cholinesterase activity of the brain of developing chick embryos. These data are presented in Table IV. The results indicate that the level of cholinesterase in the
MCOSKER
AND
TABLE Choline&erase
Activity
DANIEL
III
of Whole Blood and Blood Plasma
of Dogs
Two-tenths milliliter of heparinized whole blood or plasma was used for each assay, and duplicate determinations (a and 5) were made. Cholinesterase activity Sample No.
noles SH/lO
Whole blood 1 2 3 4 5 Blood plasma 1 2 3 5
l-
Average
b
Average
/umoles
min
SE/hr./ml.
0.130 0.125 0.126 0.110 0.135
0.130 0.130 0.123 0.111 0.138
0.130 0.128 0.125 0.111 0.137
28.80 28.13 24.98 30.83
0.143 0.145 0.153 0.191
0.155 0.155 0.153 0.199
0.149 0.150 0.153 0.195
33.53 33.75 34.43 43.88
-I
-
emymc
29.25
-
TABLE
IV Cholinesterase Activity of the Brains of Developing Chick Embryos Each sample was a composite of five brains. The tissue was removed immediately after the embryo was sacrificed by decapitation and placed in 15 ml. of ice-cold distilled water. The pooled tissue was homogenized and 3 ml. withdrawn for assay. The values represent the average of duplicates which were consistently in excellent agreement. Sample No.
Age of embryos
dL%YS
1 2 3 4 5 6 7 8 9 10 11 12
12 12 12 15 15 15 18 18 18 21 21 21
Dry weight of five brains
g. 0.12 0.12 0.11 0.25 0.26 0.25 0.44 0.46 0.45 0.54 0.53 0.50
Cholinesterase activity per g. dry tissue pm&s
161 149 181 182 173 158 219 185 191 122 241 240
per five brains SH/lO
min.
19 18 20 46 45 40 97 85 86 66 128 120
DETERMINATION
OF
i
CHOLINESTERASE
embryonic chick brain increased consistently from the 12th through the 21st day of incubation. These observations are in accord with the data of Nachmansohn (8). Agreement among the three samples within a given age group was good, except for sample 10. No plausible explanation for this low value is available. ACKNOWLEDGMENTS
The authors are most grateful to Dr. L. C. Norris for supplying the chick embryos used in this work.
and the Poultry
Department,
SUMMARY
1. *4 calorimetric micro method for the determination of cholinesterase activity using acetylthiocholine as substrate has been developed. 2. The method is rapid, requires simple laboratory equipment, and is sensitive to small amounts of enzyme; the reaction is followed by measuring the appearance of one of the reaction products (thiocholine). 3. The method has been applied to the determination of the cholinesterase activity in rat brain, in whole blood and blood plasma of dogs, and in the brains of 12-, 15-, I%, and 21-day-old chick embryos.
1. HOELLE, CT. B., J. Pharmacol. Exptl. 7’herap. 100, 158 (1950). 2. ACJGUSTINSSON, K. B., Sand. J. Clin. & Lab. Invest. ‘I, 284 (1955). 3. MEYER, A., AND WILBRANDT, W., Helv. Physiol. et Pharmacol. Acta 12, 206 (1954). 4. TABACHNICK, I. I. A., Biochiflz. et Biophys. Acta 21, 580 (1956). 5. GAL, E. M., AND ROTH, E., C/in. Chim. Acta ‘2, 316 (1957). 6. GRUNERT, R. R., AND PHILLIPS, P. H., Arch. Biochem. 30, 217 (1951). 7. SMALLMAN, B. Pu'.,AND WOLFE, L. S.,Enzymologia1’7,133 (1954). 8. NACHMANSOHN,D., Bull. sot. chim. biol. 21, 761 (1939).