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A simple and rapid chromatographic method for the immobilization of acetylcholinesterase from electric eel
ANALYTICAL
BIOCHEMISTRY
71, 273-280 (1976)
A Simple and Rapid Chromatographic Method for the Immobilization of Acetylcholinesterase from Electric Eel R. M. KOTHARI, M.A.
GRAFIUS AND D. B. MILLAR
Laboratory of Physical Biochemistry, Department of Environmental Biosciences, Naval Medical Research Institute. National Naval Medical Center, Bethesda, Maryland 20014 Received July 2, 1975: accepted September 11, 1975 Acetylcholinesterase from electric eel is selectively immobilized on Amberlite IR-120 resin equilibrated with Al 3+ ions. Immobilized acetylcholinesterase activity is stable at least for 85 days in the wet state at 10°C and for 180 days in the dry state at room temperature. Activity determinations in the presence of eserine sulfate, decamethonium bromide, quinidine sulfate and butyryl thiocholine iodide suggested that the immobilized enzyme exhibited essentially the same properties as did the free enzyme.
Various methods have been devised for the physicochemical trapping, coupling and/or adsorption of different enzymes onto water-insoluble carriers or matrices (I- 12). Using these approaches, attempts to immobilize acetylcholinesterase have resulted in stabilization of the initial enzyme activity for only a limited period (11). Although Baum et al. (5) observed retention of activity for 55 days, this was immediately followed by a rapid decline in activity. In other cases, loss of as much as 20-35% of the initial enzyme activity occurred (3,13). In certain instances, conditions for optimal enzyme coupling were complicated and multistep, involving numerous parameters (e.g., concentration of coupling agent, temperature and pH) thus rendering the procedure time consuming (10). Moreover; none of these procedures afforded a highly selective immobilization of acetylcholinesterase. Amberlite IR-120 resin equilibrated with A13+ ions has been employed for the fractionation of DNA (14), RNA (15,16), histones and other proteins (R. M. Kothari, unpublished data). The present communication describes a column chromatographic procedure, utilizing Amberlite IR-120 resin equilibrated with A13+ ions, for the preferential immobilization of acetylcholinesterase from the electric organ of the electric eel (Electrophorus electricus). The procedure is simple, rapid and easily reproducible. The immobilized enzyme is potentially useful as a field test for the detection of anticholinesterase compounds. 273 CopyrIght All rlphts
0 197h by Academ,c Press. Inc. of reproduction I” any form reserved.
274
KOTHARI,
GRAFIUS
EXPERIMENTAL
AND
MILLAR
METHODS
The ZR-220 AP column. An IR-120 A13+ complex (17) was prepared by equilibrating 10 g of dry regenerated H+ form of Amberlite IR-120 resin (Rohm and Haas Co., Philadelphia, Pa.) with 100 ml of 0.2 M AlCl, for 3 hr. Tris-HCl buffer (0.01 M, pH 8.0) was percolated through a 0.9 x 15 cm IR-120 A13+ column until the pH of the influent and effluent were the same. Extraction of acetylcholinesterase . Acetylcholinesterase was extracted at 4°C from 30 g of frozen (-20°C) electric organ tissue from E. electricus by homogenization in a Waring blender with 100 ml of 1 .O M NaCl-0.1 M Na phosphate buffer (pH 8.0) containing 1.0% Triton X-100. The homogenate was centrifuged at 78,000g for 60 min, the pellet was discarded and the supernatant was dialyzed overnight at 10°C against 10 vol of 0.01 M Tris-HCl buffer (pH 8.0). Estimation of acetylcholinesterase activity and protein content. Protein was estimated by the method of Lowry et al. (18). Protein immobilized on the column was calculated by the difference between the protein content of influent and effluent. Acetylcholinesterase activity was determined at room temperature by the DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid)) calorimetric method of Ellmanet al. (19). The following modification of the method of Ellman et al. was used to estimate the enzyme activity in the immobilized state. Three milliliters of buffer (0.1 M NaCl-0.1 M Na phosphate, pH 8.4) were added to 200 & 5 mg of water decanted, wet resin-enzyme complex, followed by 100 ~1 of 0.01 M DTNB reagent and 20 ~1 of 0.03 M acetylthiocholine iodide. The reaction mixture was then shaken on a vortex mixer for 10 set at 1 min intervals over a 5 min period. At the end of 5 min, the resin-enzyme complex was allowed to rapidly settle to the bottom of the test tube and the particlefree supernatant was transferred to a cuvette and read at 412 nm. This assay has a reproducibility of +5%. Acetylcholinesterase activity is defined as: 1 unit = 1 pmol acetylthiocholine iodide hydrolyzed/min. Immobilization of acetylcholinesterase. One hundred milliliters of crude extract of acetylcholinesterase (48% units) were centrifuged at 87OOg for 20 min at 4°C and then adsorbed on the IR-120A13+ column (0.9 x 15 cm). The column was subsequently washed with at least five bed volumes of 0.01 M Tris-HCl buffer (pH 8.0). The flow rate during adsorption and washing was 25-30 ml/hr. Fractions (5.0 ml) were collected and assayed for enzyme and protein content. The immobilized enzyme was maintained in “the wet state” by storing under the above buffer or water at 10°C. Immobilized acetylcholinesterase activity in presence of inhibitors. Eserine sulfate (Merck and Co., Inc., Rahway , N .J.), decamethonium bromide (Pfaltz and Bauer, Inc., Flushing, N. Y .), quinidine sulfate (Aldrich Chemical Co., Milwaukee, Wis.) and butyryl thiocholine iodide (Calbiochem, San Diego, Calif.) were employed to determine the acces-
IMMOBILIZED
275
ACETYLCHOLINESTERASE
sibility of the active site to inhibitors. Enzyme activity, without inhibitor, served as the control. When determining the enzyme activity in the presence of an inhibitor, 200 _+ 5 mg of wet resin-immobilized enzyme was first incubated with 3.0 ml of the inhibitor in buffer for 5 min with intermittent shaking, and then the enzyme activity was estimated as for control. RESULTS
Table 1 shows that whereas acetylcholinesterase is almost quantitatively retained on the IR-120 A13+ column, only about 2.5% of the total protein is adsorbed. An approximately 39-fold purification of the immobilized enzyme has thus beer achieved. The selective adsorption of acetylcholinesterase on the column is quite firm. Attempts to recover it by using any one of a wide variety of eluting agents under varying conditions of pH, ionic strength and temperature (Table 2) led to either partial or total loss of activity or to no elution of enzyme. The whole procedure-conversion of IR-120 H+ form into the A13+ complex form, equilibration with 0.01 M Tris-HCI buffer (pH 8.0), passage of crude extract through the column, and column washing by the same buffer to remove any loosely associated protein-required 5-6 hr. However, as IR-120 A13+ resin is stable for an indefinite period, we have been able to shorten the immobilization time by 2-3 hr by using previously prepared resin. The immobilized acetylcholinesterase was found to retain 100% of its activity, in the wet state, even after a period of 85 days (Fig. 1). Other characteristics of resin-adsorbed enzyme are: (a) wet resin exhibits activity immediately, (b) dry resin in assay medium requires a lag period of IO-60 min to give 80% of its initial immobilized activity, (c) dry resin allowed to moisten in distilled water for 1 hr before assaying shows almost the same activity as stored wet resin, and (d) immobilized enzyme TABLE IMMOBILIZATION
Total amount in the Influent Effluent Washings Matrix (retention)
OFACETYLCHOLINESTERASE
Protein (mid 510.0 412.14 84.86 13.0
1 ON THE
IR-120AP+
COLUMN
% Protein
Total enzyme units
Sp act”
% Units
loo 80.83 16.64 2.53
48% 70 0 4826
9.6 0.17 0 371
loo 1.5 0 98.5
a Specific activity is defined as: units of enzyme activity per milligram of protein.
276
KOTHARI.
GRAFIUS TABLE
AGENTS
USED
AND MILLAR 2
IN ATTEMITS TO DISSOCIATE ACETYLCHOLINESTERASE FROM THE IR-120 AP+ COLUMN
Eluting agent
Conditions of elutions
Result
1. 1.0 M NaCl-0.01 M Na phosphate buffer (with and without 1.0% Triton X-100)
pH 6.0, 8.0
No elutiot?
2. 1.0 M NaCl-0.5
pH 7.0
Loss of activity*
3. 1.0 M ammonium acetate
pH 7.0
No elution
4. 1.O M ammonium chloride
-
No elution
5. Na phosphate buffer alone
0.01-0.5 M pH6.0-8.0
No elution with loss of activity at high ionic strength
6. 0.1 M Tris-HCl-0.01 MgCI, buffer
pH 6.0, 8.0
No elution
0.5, 1.0 M
No elution
8. Lysine-HCl
1.0 M
No elution
9. EDTA
0.1, 0.5, 1.0 M pH 7.0
No elution
10. Distilled water
Repeated freezing and thawing
No elution
Il. Increasing temperature in 0.1 M NaCl
30, 40, 50°C
Total loss of activity at 50°C
M
Na
phosphate buffer
M
7. Magnesium chloride
’ No elution means absence of enzyme activity in the supernatant, but its presence on the resin. ’ Loss of activity means the reduction of detectable enzyme activity both in the supematant and on the resin.
on resin is quite stable at room temperature (ca. 27°C) in the dry state for a period of at least 6 months (70% of initial adsorbed activity). Studies on the feasibility of repeated use of immobilized enzyme have revealed that the same sample of resin-immobilized enzyme activity can be used at least 60-100 times with 100% efficiency in the first 40 activity determinations and with 80% efficiency in subsequent determinations (Fig. 2). It is unlikely that water-induced leaching of the enzyme from the matrix can account for these findings since washing of an IR-120 A13+ column with 5 liters of distilled water did not detectably affect the activity of the adsorbed enzyme. Activity determinations using wet resin-immobilized enzyme in the presence of inhibitors showed that 3.2 X lo-’ M eserine sulfate, and
IMMOBILIZED
ACETYLCHOLINESTERASE
277
FIG. 1. Stability of adsorbed enzyme. Approximately 10 g of resin-enzyme complex was placed in about 100 ml distilled water and stored in a covered beaker at 10°C. For assay, a 200 2 5 mg aliquot was removed and assayed as described in the text. The values shown are averages of 3-4 assays (precision < 5%).
8.0 x lop7 M decamethonium bromide inhibited 87 and 77% of the initial activity, respectively (Fig. 3). Quinidine sulfate (5 x 10e3 M) also inhibited the enzyme by 31%. Activity determinations of equivalent amounts of free enzyme under identical inhibitor conditions showed 100,95 and 33% inhibition of the initial activity by eserine sulfate, decamethonium bromide and quinidine sulfate, respectively. Immobilized and free enzyme did not hydrolyze detectable amounts of butyryl thiocholine iodide. The integrity of butyryl thiocholine iodide was checked with authentic butyrylcholinesterase. DISCUSSION
Conditions have been devised for optimal and preferential adsorption of acetylcholinesterase on an IR-120 A13+ column. The adsorbed enzyme is stable for at least 6 months. At present, no effective conditions have been devised for the elution of the adsorbed acetylcholinesterase. If such conditions can be found, the present system of selective immobilization on an IR-120 A13+ column could furnish a single step procedure for its purification. The lack of success of a variety of attempts to elute the enzyme (Table 2) suggest the binding forces to be firm and complex. The properties of IR-120 A13+ that make it a convenient and promising tool for immobilization of acetylcholinesterase are an easy, rapid, and simple method of preparation, a good flow rate and an absence of clogging. The very similar inhibition of free and immobilized enzyme by several inhibitors showed that access of the inhibitor to the active site of the adsorbed enzyme was not hindered by some matrix-induced constraint on the enzyme. The inhibitor incubation requirement may reflect the time
278
KOTHARI,
GRAFIUS
AND MILLAR
FIG. 2. Effects of repeated assay of adsorbed enzyme. A 200 2 5 mg aliquot of resinenzyme complex was employed and the enzyme assay was performed as described in the text. At the completion of each assay, the resin-enzyme complex was washed twice with 10 ml distilled water and then the decanted resin-enzyme complex was reassayed. About 20 assays were done consecutively each day. At the end of 20 assays, the resin-enzyme complex was again washed twice with distilled water and stored under 10 ml water at 10°C. The following day, a further 20 assays were performed as indicated.
needed for the bulkier inhibitor to diffuse into the matrix. Additionally, matrix binding induced conformational alterations in the enzyme may be partially responsible. The problem is clearly complex. The 3 1% inhibition of enzyme activity by 5 x 1O-3 M quinidine sulfate is in agreement with the observations of Wright and Sabine (20) that quinidine sulfate inhibited human red blood cell acetylcholinesterase less effectively than plasma cholinesterase. No detectable rate of hydrolysis of butyryl thiocholine iodide by the immobilized as well as free enzyme compared to its high rate of hydrolysis by plasma butyryl cholinesterase confirmed that we were dealing with a true acetylcholinesterase (20). These studies showed that immobilized enzyme exhibits essentially the same properties as does free enzyme. Immobilized acetylcholinesterase was stable in the wet state at 10°C for at least 85 days and in the dry state at 27°C for at least 180 days. These studies suggest that acetylcholinesterase immobilized on IR-120A13+ resin has the potential of being repeatedly used as an assay tool to determine the amount of acetylcholine in biological fluids. Further, preliminary studies indicate that a simple calorimetric enzymatic assay involving the use of pH range finding indicators can be quantitated to detect the presence of pesticides and other anticholinesterase agents by virtue of their inhibition of the immobilized enzyme.
IMMOBILIZED
INHIBITOR
ACETYLCHOLINESTERASE
CONCENTRATION
279
(Mx IO’)
FIG. 3. Effect of preincubation of acetylcholinesterase inhibitors on adsorbed enzyme. Preincubation of the enzyme with inhibitor, as described in the text, was necessary since if the inhibitor was added along with the substrate, little or no inhibition was observed. Preincubation and activity assay were performed at room temperature. (0) Eserine sulfate; (0) decamethonium bromide.
ACKNOWLEDGMENT R. M. Kothari is a Postdoctoral Research Associate of the National Research Council and National Academy of Sciences. From the Bureau of Medicine and Surgery, Navy Department Research Subtasks MR000.01.01.1084 and MF51.524.014.9025. The opinions and statements contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Silman, I. H., and Katchalsky, E. (1%6) Annu. Rev. Biochem. 35,873. Goldstein, L., and Katchalsky, E. (1%8) Fresenius’ Z. Anal. Chem. 243, 375. Axen, R., Heilbronn, E., and Winter, A. (1969) Biochim. Biophys. Acta 191, 478. Mosbach, K. (1970)Acta Chem. Scnnd. 24, 2084. Baum, G., Ward, F. B., and Weetall, H. H. (1972) Biochim. Biophys. Acra 268, 411. Orth, H. D., and Brtimmer, W. (1972)Angew. Chem. 84, 319. Brtimmer, W., Hennrich, N., Klockow, M.. Lang, H.. and Orth, H. D. (1972) Eur. J. Biochem. 25, 129. Bunting, P. S., and Laidler, K. J. (1972) Biochemistry 11, 4477. Turkova, J., Hubalkova, O., Kiivakova, M., and Coupek, J. (1973) Biochim. Biophys. Acta 322, 1. Johansson. A. C., and Mosbach, K. (1974) Biochim. Biophys. Acra 370, 339, 348. Alsen, C., Bertram, U., Gersteuer. T., Ohnesorge, F. K., and Delin, S. (1975) Biochim. Biophys. Acta 377, 297. Ngo, T. T., and Laidler. K. J. (1975) Biochim. Biophys. Acta 377, 303. Guilbault, G. G., and Das, J. (1970)Anal. Biochem. 33, 341. Kothari, R. M. (1970) Chromatogr. Rev. 12, 127. Kothari. R. M. (1971)J. Chromatogr. 57, 83.
280
KOTHARI.
GRAFIUS
AND MILLAR
16. Shankar, V., and Joshi, P. N. (1974) J. Chromatogr. 90,99. 17. Kothari, R. M. (1970) J. Chromarogr. 52, 119. 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 19. Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961) Biothem.
Pharmacol.
I, 88.
20. Wright, C. I., and Sabine, J. C. (1948) J. Pharmacol.
Exp.
Thu.
93, 230.
BIOCHEMISTRY
71, 273-280 (1976)
A Simple and Rapid Chromatographic Method for the Immobilization of Acetylcholinesterase from Electric Eel R. M. KOTHARI, M.A.
GRAFIUS AND D. B. MILLAR
Laboratory of Physical Biochemistry, Department of Environmental Biosciences, Naval Medical Research Institute. National Naval Medical Center, Bethesda, Maryland 20014 Received July 2, 1975: accepted September 11, 1975 Acetylcholinesterase from electric eel is selectively immobilized on Amberlite IR-120 resin equilibrated with Al 3+ ions. Immobilized acetylcholinesterase activity is stable at least for 85 days in the wet state at 10°C and for 180 days in the dry state at room temperature. Activity determinations in the presence of eserine sulfate, decamethonium bromide, quinidine sulfate and butyryl thiocholine iodide suggested that the immobilized enzyme exhibited essentially the same properties as did the free enzyme.
Various methods have been devised for the physicochemical trapping, coupling and/or adsorption of different enzymes onto water-insoluble carriers or matrices (I- 12). Using these approaches, attempts to immobilize acetylcholinesterase have resulted in stabilization of the initial enzyme activity for only a limited period (11). Although Baum et al. (5) observed retention of activity for 55 days, this was immediately followed by a rapid decline in activity. In other cases, loss of as much as 20-35% of the initial enzyme activity occurred (3,13). In certain instances, conditions for optimal enzyme coupling were complicated and multistep, involving numerous parameters (e.g., concentration of coupling agent, temperature and pH) thus rendering the procedure time consuming (10). Moreover; none of these procedures afforded a highly selective immobilization of acetylcholinesterase. Amberlite IR-120 resin equilibrated with A13+ ions has been employed for the fractionation of DNA (14), RNA (15,16), histones and other proteins (R. M. Kothari, unpublished data). The present communication describes a column chromatographic procedure, utilizing Amberlite IR-120 resin equilibrated with A13+ ions, for the preferential immobilization of acetylcholinesterase from the electric organ of the electric eel (Electrophorus electricus). The procedure is simple, rapid and easily reproducible. The immobilized enzyme is potentially useful as a field test for the detection of anticholinesterase compounds. 273 CopyrIght All rlphts
0 197h by Academ,c Press. Inc. of reproduction I” any form reserved.
274
KOTHARI,
GRAFIUS
EXPERIMENTAL
AND
MILLAR
METHODS
The ZR-220 AP column. An IR-120 A13+ complex (17) was prepared by equilibrating 10 g of dry regenerated H+ form of Amberlite IR-120 resin (Rohm and Haas Co., Philadelphia, Pa.) with 100 ml of 0.2 M AlCl, for 3 hr. Tris-HCl buffer (0.01 M, pH 8.0) was percolated through a 0.9 x 15 cm IR-120 A13+ column until the pH of the influent and effluent were the same. Extraction of acetylcholinesterase . Acetylcholinesterase was extracted at 4°C from 30 g of frozen (-20°C) electric organ tissue from E. electricus by homogenization in a Waring blender with 100 ml of 1 .O M NaCl-0.1 M Na phosphate buffer (pH 8.0) containing 1.0% Triton X-100. The homogenate was centrifuged at 78,000g for 60 min, the pellet was discarded and the supernatant was dialyzed overnight at 10°C against 10 vol of 0.01 M Tris-HCl buffer (pH 8.0). Estimation of acetylcholinesterase activity and protein content. Protein was estimated by the method of Lowry et al. (18). Protein immobilized on the column was calculated by the difference between the protein content of influent and effluent. Acetylcholinesterase activity was determined at room temperature by the DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid)) calorimetric method of Ellmanet al. (19). The following modification of the method of Ellman et al. was used to estimate the enzyme activity in the immobilized state. Three milliliters of buffer (0.1 M NaCl-0.1 M Na phosphate, pH 8.4) were added to 200 & 5 mg of water decanted, wet resin-enzyme complex, followed by 100 ~1 of 0.01 M DTNB reagent and 20 ~1 of 0.03 M acetylthiocholine iodide. The reaction mixture was then shaken on a vortex mixer for 10 set at 1 min intervals over a 5 min period. At the end of 5 min, the resin-enzyme complex was allowed to rapidly settle to the bottom of the test tube and the particlefree supernatant was transferred to a cuvette and read at 412 nm. This assay has a reproducibility of +5%. Acetylcholinesterase activity is defined as: 1 unit = 1 pmol acetylthiocholine iodide hydrolyzed/min. Immobilization of acetylcholinesterase. One hundred milliliters of crude extract of acetylcholinesterase (48% units) were centrifuged at 87OOg for 20 min at 4°C and then adsorbed on the IR-120A13+ column (0.9 x 15 cm). The column was subsequently washed with at least five bed volumes of 0.01 M Tris-HCl buffer (pH 8.0). The flow rate during adsorption and washing was 25-30 ml/hr. Fractions (5.0 ml) were collected and assayed for enzyme and protein content. The immobilized enzyme was maintained in “the wet state” by storing under the above buffer or water at 10°C. Immobilized acetylcholinesterase activity in presence of inhibitors. Eserine sulfate (Merck and Co., Inc., Rahway , N .J.), decamethonium bromide (Pfaltz and Bauer, Inc., Flushing, N. Y .), quinidine sulfate (Aldrich Chemical Co., Milwaukee, Wis.) and butyryl thiocholine iodide (Calbiochem, San Diego, Calif.) were employed to determine the acces-
IMMOBILIZED
275
ACETYLCHOLINESTERASE
sibility of the active site to inhibitors. Enzyme activity, without inhibitor, served as the control. When determining the enzyme activity in the presence of an inhibitor, 200 _+ 5 mg of wet resin-immobilized enzyme was first incubated with 3.0 ml of the inhibitor in buffer for 5 min with intermittent shaking, and then the enzyme activity was estimated as for control. RESULTS
Table 1 shows that whereas acetylcholinesterase is almost quantitatively retained on the IR-120 A13+ column, only about 2.5% of the total protein is adsorbed. An approximately 39-fold purification of the immobilized enzyme has thus beer achieved. The selective adsorption of acetylcholinesterase on the column is quite firm. Attempts to recover it by using any one of a wide variety of eluting agents under varying conditions of pH, ionic strength and temperature (Table 2) led to either partial or total loss of activity or to no elution of enzyme. The whole procedure-conversion of IR-120 H+ form into the A13+ complex form, equilibration with 0.01 M Tris-HCI buffer (pH 8.0), passage of crude extract through the column, and column washing by the same buffer to remove any loosely associated protein-required 5-6 hr. However, as IR-120 A13+ resin is stable for an indefinite period, we have been able to shorten the immobilization time by 2-3 hr by using previously prepared resin. The immobilized acetylcholinesterase was found to retain 100% of its activity, in the wet state, even after a period of 85 days (Fig. 1). Other characteristics of resin-adsorbed enzyme are: (a) wet resin exhibits activity immediately, (b) dry resin in assay medium requires a lag period of IO-60 min to give 80% of its initial immobilized activity, (c) dry resin allowed to moisten in distilled water for 1 hr before assaying shows almost the same activity as stored wet resin, and (d) immobilized enzyme TABLE IMMOBILIZATION
Total amount in the Influent Effluent Washings Matrix (retention)
OFACETYLCHOLINESTERASE
Protein (mid 510.0 412.14 84.86 13.0
1 ON THE
IR-120AP+
COLUMN
% Protein
Total enzyme units
Sp act”
% Units
loo 80.83 16.64 2.53
48% 70 0 4826
9.6 0.17 0 371
loo 1.5 0 98.5
a Specific activity is defined as: units of enzyme activity per milligram of protein.
276
KOTHARI.
GRAFIUS TABLE
AGENTS
USED
AND MILLAR 2
IN ATTEMITS TO DISSOCIATE ACETYLCHOLINESTERASE FROM THE IR-120 AP+ COLUMN
Eluting agent
Conditions of elutions
Result
1. 1.0 M NaCl-0.01 M Na phosphate buffer (with and without 1.0% Triton X-100)
pH 6.0, 8.0
No elutiot?
2. 1.0 M NaCl-0.5
pH 7.0
Loss of activity*
3. 1.0 M ammonium acetate
pH 7.0
No elution
4. 1.O M ammonium chloride
-
No elution
5. Na phosphate buffer alone
0.01-0.5 M pH6.0-8.0
No elution with loss of activity at high ionic strength
6. 0.1 M Tris-HCl-0.01 MgCI, buffer
pH 6.0, 8.0
No elution
0.5, 1.0 M
No elution
8. Lysine-HCl
1.0 M
No elution
9. EDTA
0.1, 0.5, 1.0 M pH 7.0
No elution
10. Distilled water
Repeated freezing and thawing
No elution
Il. Increasing temperature in 0.1 M NaCl
30, 40, 50°C
Total loss of activity at 50°C
M
Na
phosphate buffer
M
7. Magnesium chloride
’ No elution means absence of enzyme activity in the supernatant, but its presence on the resin. ’ Loss of activity means the reduction of detectable enzyme activity both in the supematant and on the resin.
on resin is quite stable at room temperature (ca. 27°C) in the dry state for a period of at least 6 months (70% of initial adsorbed activity). Studies on the feasibility of repeated use of immobilized enzyme have revealed that the same sample of resin-immobilized enzyme activity can be used at least 60-100 times with 100% efficiency in the first 40 activity determinations and with 80% efficiency in subsequent determinations (Fig. 2). It is unlikely that water-induced leaching of the enzyme from the matrix can account for these findings since washing of an IR-120 A13+ column with 5 liters of distilled water did not detectably affect the activity of the adsorbed enzyme. Activity determinations using wet resin-immobilized enzyme in the presence of inhibitors showed that 3.2 X lo-’ M eserine sulfate, and
IMMOBILIZED
ACETYLCHOLINESTERASE
277
FIG. 1. Stability of adsorbed enzyme. Approximately 10 g of resin-enzyme complex was placed in about 100 ml distilled water and stored in a covered beaker at 10°C. For assay, a 200 2 5 mg aliquot was removed and assayed as described in the text. The values shown are averages of 3-4 assays (precision < 5%).
8.0 x lop7 M decamethonium bromide inhibited 87 and 77% of the initial activity, respectively (Fig. 3). Quinidine sulfate (5 x 10e3 M) also inhibited the enzyme by 31%. Activity determinations of equivalent amounts of free enzyme under identical inhibitor conditions showed 100,95 and 33% inhibition of the initial activity by eserine sulfate, decamethonium bromide and quinidine sulfate, respectively. Immobilized and free enzyme did not hydrolyze detectable amounts of butyryl thiocholine iodide. The integrity of butyryl thiocholine iodide was checked with authentic butyrylcholinesterase. DISCUSSION
Conditions have been devised for optimal and preferential adsorption of acetylcholinesterase on an IR-120 A13+ column. The adsorbed enzyme is stable for at least 6 months. At present, no effective conditions have been devised for the elution of the adsorbed acetylcholinesterase. If such conditions can be found, the present system of selective immobilization on an IR-120 A13+ column could furnish a single step procedure for its purification. The lack of success of a variety of attempts to elute the enzyme (Table 2) suggest the binding forces to be firm and complex. The properties of IR-120 A13+ that make it a convenient and promising tool for immobilization of acetylcholinesterase are an easy, rapid, and simple method of preparation, a good flow rate and an absence of clogging. The very similar inhibition of free and immobilized enzyme by several inhibitors showed that access of the inhibitor to the active site of the adsorbed enzyme was not hindered by some matrix-induced constraint on the enzyme. The inhibitor incubation requirement may reflect the time
278
KOTHARI,
GRAFIUS
AND MILLAR
FIG. 2. Effects of repeated assay of adsorbed enzyme. A 200 2 5 mg aliquot of resinenzyme complex was employed and the enzyme assay was performed as described in the text. At the completion of each assay, the resin-enzyme complex was washed twice with 10 ml distilled water and then the decanted resin-enzyme complex was reassayed. About 20 assays were done consecutively each day. At the end of 20 assays, the resin-enzyme complex was again washed twice with distilled water and stored under 10 ml water at 10°C. The following day, a further 20 assays were performed as indicated.
needed for the bulkier inhibitor to diffuse into the matrix. Additionally, matrix binding induced conformational alterations in the enzyme may be partially responsible. The problem is clearly complex. The 3 1% inhibition of enzyme activity by 5 x 1O-3 M quinidine sulfate is in agreement with the observations of Wright and Sabine (20) that quinidine sulfate inhibited human red blood cell acetylcholinesterase less effectively than plasma cholinesterase. No detectable rate of hydrolysis of butyryl thiocholine iodide by the immobilized as well as free enzyme compared to its high rate of hydrolysis by plasma butyryl cholinesterase confirmed that we were dealing with a true acetylcholinesterase (20). These studies showed that immobilized enzyme exhibits essentially the same properties as does free enzyme. Immobilized acetylcholinesterase was stable in the wet state at 10°C for at least 85 days and in the dry state at 27°C for at least 180 days. These studies suggest that acetylcholinesterase immobilized on IR-120A13+ resin has the potential of being repeatedly used as an assay tool to determine the amount of acetylcholine in biological fluids. Further, preliminary studies indicate that a simple calorimetric enzymatic assay involving the use of pH range finding indicators can be quantitated to detect the presence of pesticides and other anticholinesterase agents by virtue of their inhibition of the immobilized enzyme.
IMMOBILIZED
INHIBITOR
ACETYLCHOLINESTERASE
CONCENTRATION
279
(Mx IO’)
FIG. 3. Effect of preincubation of acetylcholinesterase inhibitors on adsorbed enzyme. Preincubation of the enzyme with inhibitor, as described in the text, was necessary since if the inhibitor was added along with the substrate, little or no inhibition was observed. Preincubation and activity assay were performed at room temperature. (0) Eserine sulfate; (0) decamethonium bromide.
ACKNOWLEDGMENT R. M. Kothari is a Postdoctoral Research Associate of the National Research Council and National Academy of Sciences. From the Bureau of Medicine and Surgery, Navy Department Research Subtasks MR000.01.01.1084 and MF51.524.014.9025. The opinions and statements contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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