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New procedure for rapid and sensitive detection of cholinesterase separated by polyacrylamide gel electrophoresis
New
Procedure Cholinesterase
for
Rapid
Separated Gel
G. G. GUILBAULT, Department
and
Sensitive
Detection
of
by Polyacrylamide
Electrophoresis
S. S. KII’AN!
J. TULLY,
AND
of Chemistry, Louisiana Stale Univei:siiy Lakefront Campus, New Orleans, L0u.i.G~~
Received November
I). HACKNEY in SW 701%
C)I.~CIIILS,
21, 1969
Several histochemical methods have been established for the identification of choline&erase (ChE) in animal &sues (l-5) ; these methods have been suggested for the detection of ChE after electrophoresis (6). However, attempts in our laboratory to employ these methods for the demonstration of ChE after polyacrylamide gel electrophoresis indicated long incubation times are required for the development of enzyme bands in the gel. Furthermore, only i)rcl,at”at,ions of high c~nzyrne activity could bc detected due t,o the low sensitiyit.y of these substrates, and the resolving power is diminished becauw of the spreading of the fractionated enzyme bands during incubation. The lack of sensitivity of other methods makes them very difficult to scale clown to micro level. Recently, Guilbault and Eadar described the use of N-methyl indoxyl acetate (NMIA) as sub&ate for the sensitive determination of cholinesterase (7). We have wetI this substrate for tlw micro detection of ChE in gels after c~lect~rophoresi~. Thr new method7 which is presented here, is very rapid, simple, and selwiti\.(i. MBTERIALS
1. N-methyl indoxyl acetate: prepared according tmo the method of Guilbault ef nE. (7) (available from Isolab, Inc., Elkhart, Indiana). 2. Horse serum ChE (available from Sigma Chemicals, St. Louis, Missouri). 3. Sheep and chicken liver ChE: prepared according to the method of Guilbault et al. (8). 4. Fire ant and cockroach ChE: prepared according to the method of Guilkault et ul. (9). 5. Lipase (available from Calbiochem Corp., Los Angeles, California). 6. Cellulase (available from Sigma Chemicals, St. Louis, ‘Missouri), 72
DETECTION
IN
POLYACRYL.4MIDE
GELS
73
CHOLINESTERASE
METHODS
Electrophoresis in polyacrylamide gels wa,s accomplished essentially as described by Davis (lo), employing 7% polyacrylamide in the running gel at pB 8.0 and using bromophenol blue as tracking dye. Gels were cast in glass tubes 90 cm in length and 5 mm inside diameter. Approximately 200 ~1 of sample gel solution was layered above the spacegel. A constant current of 2.5 mA was applied to each t’ube until the dye front had migrated to about 10 mm from the end. After the completion of electrophoresis, the gel cylinders were rinsed well with distilled water and then placed in a test tube containing incubating solut’ion.Test tubes were then incubated in a suitable water bath at 37°C. After the complete development of visualized enzyme bands, the gel was again rinsed with distilled water, placed in distilled water for IO minutes and than transferred into 0.1 M, pH 7.0, phosphate buffer solution for photographing or preserva.tion. Several incubating solutions were designed in an attempt to obtain better results for this detection and were prepared about 1 hour before use. These are given in Table 1. Clomposit,ion
Soln. 1 2 3 4 5 6 7
of 1ncubat’in.g Solutions
TABLE I for Detection
Type and amt. of 0.1 M pH 7.5 buffer soln. nsed 25 25 25 25 25 25
ml ml ml ml ml ml
0.5 gm NMIA in
Tris phos Tris phos Tris phos
1 ml 1 ml 3 ml 3 ml 5 ml 5 ml 25 ml
‘I Tris = 2-amino-2-hydroxymet.hyl-1,Spropanediol; RESULTS
Sensitivity
of ChE in Disc Elect.rophores:+i
AND
Triply distilled water added
dioxane dioxane ethanol ethanol dist,d. H&1 distd. Hz0 distd. Hz0
24 24 22 22 20 20 25
ml ml ml ml ml ml ml
phoe = phosphate.
DISCUSSION
and Substrate Specificity
Since the sensitivity of the enzyme detection in the gel dependsmostly on the easeof substrate penetration and the rate of substrate hydrolysis, more than IO substrates have been tried for their sensitivity against ChE in this experiment. These substrates included naphthyl esters, indoxyl esters and their substitute analogs, indophenyl acetate, etc. As judged by the rate of reactivity as well as the stability and precise location of
wzyme band ii1 t.he zyniograni. N h’l l,L\,was fonnci to l)e the best su1tstrat.e. A zymogram rc5ulLing from licrr~c~wr~m ChE t cr~v:ird t,his substrate i5 shown in Fig. 1. As cm 1~ well ii-0m this figures. tlw applied enzyme as low as 0.01 unit per t’ubc coul(.l bc detected. This i,+ especially useful for evaluating preparations at the low ChE level found in insects. 3loreover, the use of NMIA has several ad\x~tagos over otlier sulx$rates: (.I) The react’ion is very rapid. Clex c~~~zyrnc bandt; de\-eloped within a few minutes after the addit,ion of the incubatjing solut’ion when the applied enzyme was I unit per t’ube or more. (2) The colorless substrate is cnzymically hydrolyzed and converted into a greenish material (see II or III below) which cm be easily recognized by its int’ense color. (3) In the presence of oxygen, t,he enzymically hydrolyzed mat,erial I$‘-methyl indoxyl (IIj dimerized automatically and the final product is a highly insoluble greenish precipitate (,IV) (7) ; no coupling reagent such as diazonium, potassium cyanide, copper sulfide, etc. is required. The overall reaction is shown in Scheme I, As previously reported by Guilbault et aE. (7), NMIA is not specific,
3
1.
0.5
0.1
-
ChE Bands
-
Dye front
0.01 Units
of
ChE
FIG. 1. Electrophoretic patterns of horse serum ChE obtained by incubating with N-methyl indoxyl acetate. Enzyme concentrations range from 3 to 0.01 unit (units are expressed as pmoles acetylcholine chloride hydrolyzed per minute at 25°C and pH 7.4). Picture taken 5 minutes after incubation. Tucubation solution (6).
DETECTION
L N-Methyl Acetate
Indoxyt
IN
POLYACRYLAMIDE
GELS
11 N-Methyl Indoxyl Green
CWOLINE!STERASE
75
III N-Methyl Indoxyl Green
IV N-Methyl indigo Green SCHEME
I
but is hydrolyzed by lipase and cellulase, Substrate specificity towards both enzymes has been tested and our results indicated t)hat lipase had very low reactivity against this substrate in the gel. UsuaIly it took more than 12 hours to obtain clear enzyme bands. In this trial cellulase was almost inactive since no color band was observed, This may be due either to the inactivation of this enzyme during elect,rophoresis,or to the low reactivity of this enzyme toward the substrate. The activity of ChE from different sources toward this substrate has also been tested. In general, sharp enzyme bands of high color intensit’y were always obtained with insect ChE and widespread enzyme bands were observed with serum and liver ChE. Our previous studies on the molecular size estimation for these partially purified enzymes on SsephadexG-100 indicated that insect ChE appeared to be larger molecules than those of liver and serum ChE. The specific activity of the insect ChE used in this experiment was also lower compared with liver and serum CHE, It appears, therefore, that the larger insect ChE molecule might have slowed down the rapid diffusion of the enzyme in the gel after electrophoresis since the specificity of insect ChE used in this experiment was much lower t’han that of liver and serum ChE. EfJect of Incubatin-g System
Even though NMIA is much more soluble in systems containing organic solvents, only slight differences were obtained from systems with and without the addition of organic solvents. Generally speaking, the development of color enzyme bands was faster, and the precipitate formed in the gel was finer, in systems with organic solvents. This may be due to the
fact’ that tlw organis so1vcnt.s t~cill) tv bring more wbstrate ink) t,he incubating syst’em, and this high ;;ubstrate concc?nt,ration accelerated &he rate of penetration and reactioll. I-Iowevcr, the gradual development’ of a greenish background in noncnzylnc~ areas tended to obscure the exact localization of t’he enzyme hand. This interference became a serious problem when samples contained v(lry lmowenzyme activity. It is believed that the formation of the greenish background WRY clue to the nonenzymic hydrolysis of the substrate since a gradual appearance of a light. g,reenish color was also found after the expo.Gurc of blank gels, without any addition of ChE, in the incubating solutions for the samt’ period of t’ime. This same type of behavior was observed in homogeneous solution (7). The effect of different buffers was not st’riking. Good results could be obtained with both buffers, but a IiMe greenish background could also be found on long standing. For the detectZion of preparations containing extremely low activity, System 7 is recommrnded even though much longer incubating time is required. Distinctj enzyme bancis with clear background were a.lways obtained in t,his system after a 10 hour incubation. Effect of Substrnte and Freshness of Medin Different lots of substrate were found to give different results, e.g., the newer the substrate the faster the reaction. It, was also found that the fresher the incubating media, the more rapid is the reaction taking place in the gel. Thus, to obtain best. results, the time hctween media preparation and the start of incubation should be kept minimum.
When the volume ratio of the gel freshly removed from the electrophoresis tube to the incubating solution was lj the enzyme band showed up very slowly and the band int.ensity was faint. The rat’e of color development as well as the color int,ensity increased as the volume of the incubating solution increased. The slow development of color enzyme band was possibly due to the presence of small amounts ,of the substrate in the incubating solution and this limited amount of the substrate was insufficient for optimum reaction in the gel. In addition, the leakage of the enzyme out of gel might, have occurred during long incubation times since t.he condition was unfavorable for prompt enzyme rclaction. -4 faint color enzyme band was thus obtained. For best results, the volume of incubating solution should be at least 5 times that of the gel volume.
Enzyme bands were rapidly dccolorized when the g,el was stored in either acidic or alkaline saluCirrn, Prolmonged storage of gels in distilled
DETECTION
IN
POLYACRYLAMIDE
GELS
CHOLIKESTERASE
77
water also led to a decrease in the band intensity and the gel pattern ultimately faded. However, the rate of fading may be reduced by storage of gels in either Tris or phosphate buffer at 0.1 M, pH 7.0. No dramatic change in band intensily was noted within 20 days. SUMMARY
A convenient method for the rapid and precise dekction of cholinesterase (ChE) in polyacrylamide gels is described. A vastSly increased sensitivity was achieved by the use of N-methyl indoxyl acetat’e as substrate. As low as 1 x IO-” unit ChE can be detected within a few minutes by simply immersing the gel cylinder in the incubat,ing mixture. X:0 coupling reagent is required. ACKNOWLEDGMENT The financial Health Service, acknowledged.
supportof the Department NationaI
Institutes
of Health. Education and Welfare, Pub’i~ of Health, Grant ES 0042601, is gratefuli)
REFEREXCES 1. BARKA, T., AND ANDERSON, P. J. “Histochemistry, Theory, Practice and Bibliography,” p, 203. Hoeber (Harper & Row), New York, 1963. 2. BUR.STONE,M. S., J. N&l. Cancer Inst. 18, 167 (1957). 3. PEARSON,B., AND GROSE,F., A. M. A. Arch. Path&g. 67, 324 (1959). 4. SAVAY, GY., AND CSILLIK, B., Acta H&to&em. 6, 307 (1959). 5. GEREBTZOFF,M. A., Acta Anat. 19, 366 (1953). 6. ZWEIG, G., AND WHITAKER, J. R., “Paper Chromatography and Electrophoresis,” Vol. I, p. 176. Academic Press, New York. 1967. 7. GUILBAWLT, G. G., SADAR, M. H.. GLMER, R., AND SKOU. C.. Anal. Letters 1, 365 (1968). 8. GUILBAULT, G. G., SADAR,M. H., KAUN, S. S.. AND CASEY, D.. “The E&et of Pesticides on Liver Cholinesterases from Rabbit, Pigeon, Chicken, Sheep and Pig,” Anal, Chim. Acta, in press. 9. GUILB.~ULT, G. G., KUAN, S. S., AND SADAR,M. H., “Purification and Properties of Cholinesterases from Honey Bees-Apti millifera Linnaeus and Boll WeevilsAnthonamus grandk Boheman,” J. Agr. Food Chem., in press. 10. DAVIS, B. J., “Disc Electrophoresis,” Ann. N. Y. Acad. Sci. 121, 404 (1964).
Procedure Cholinesterase
for
Rapid
Separated Gel
G. G. GUILBAULT, Department
and
Sensitive
Detection
of
by Polyacrylamide
Electrophoresis
S. S. KII’AN!
J. TULLY,
AND
of Chemistry, Louisiana Stale Univei:siiy Lakefront Campus, New Orleans, L0u.i.G~~
Received November
I). HACKNEY in SW 701%
C)I.~CIIILS,
21, 1969
Several histochemical methods have been established for the identification of choline&erase (ChE) in animal &sues (l-5) ; these methods have been suggested for the detection of ChE after electrophoresis (6). However, attempts in our laboratory to employ these methods for the demonstration of ChE after polyacrylamide gel electrophoresis indicated long incubation times are required for the development of enzyme bands in the gel. Furthermore, only i)rcl,at”at,ions of high c~nzyrne activity could bc detected due t,o the low sensitiyit.y of these substrates, and the resolving power is diminished becauw of the spreading of the fractionated enzyme bands during incubation. The lack of sensitivity of other methods makes them very difficult to scale clown to micro level. Recently, Guilbault and Eadar described the use of N-methyl indoxyl acetate (NMIA) as sub&ate for the sensitive determination of cholinesterase (7). We have wetI this substrate for tlw micro detection of ChE in gels after c~lect~rophoresi~. Thr new method7 which is presented here, is very rapid, simple, and selwiti\.(i. MBTERIALS
1. N-methyl indoxyl acetate: prepared according tmo the method of Guilbault ef nE. (7) (available from Isolab, Inc., Elkhart, Indiana). 2. Horse serum ChE (available from Sigma Chemicals, St. Louis, Missouri). 3. Sheep and chicken liver ChE: prepared according to the method of Guilbault et al. (8). 4. Fire ant and cockroach ChE: prepared according to the method of Guilkault et ul. (9). 5. Lipase (available from Calbiochem Corp., Los Angeles, California). 6. Cellulase (available from Sigma Chemicals, St. Louis, ‘Missouri), 72
DETECTION
IN
POLYACRYL.4MIDE
GELS
73
CHOLINESTERASE
METHODS
Electrophoresis in polyacrylamide gels wa,s accomplished essentially as described by Davis (lo), employing 7% polyacrylamide in the running gel at pB 8.0 and using bromophenol blue as tracking dye. Gels were cast in glass tubes 90 cm in length and 5 mm inside diameter. Approximately 200 ~1 of sample gel solution was layered above the spacegel. A constant current of 2.5 mA was applied to each t’ube until the dye front had migrated to about 10 mm from the end. After the completion of electrophoresis, the gel cylinders were rinsed well with distilled water and then placed in a test tube containing incubating solut’ion.Test tubes were then incubated in a suitable water bath at 37°C. After the complete development of visualized enzyme bands, the gel was again rinsed with distilled water, placed in distilled water for IO minutes and than transferred into 0.1 M, pH 7.0, phosphate buffer solution for photographing or preserva.tion. Several incubating solutions were designed in an attempt to obtain better results for this detection and were prepared about 1 hour before use. These are given in Table 1. Clomposit,ion
Soln. 1 2 3 4 5 6 7
of 1ncubat’in.g Solutions
TABLE I for Detection
Type and amt. of 0.1 M pH 7.5 buffer soln. nsed 25 25 25 25 25 25
ml ml ml ml ml ml
0.5 gm NMIA in
Tris phos Tris phos Tris phos
1 ml 1 ml 3 ml 3 ml 5 ml 5 ml 25 ml
‘I Tris = 2-amino-2-hydroxymet.hyl-1,Spropanediol; RESULTS
Sensitivity
of ChE in Disc Elect.rophores:+i
AND
Triply distilled water added
dioxane dioxane ethanol ethanol dist,d. H&1 distd. Hz0 distd. Hz0
24 24 22 22 20 20 25
ml ml ml ml ml ml ml
phoe = phosphate.
DISCUSSION
and Substrate Specificity
Since the sensitivity of the enzyme detection in the gel dependsmostly on the easeof substrate penetration and the rate of substrate hydrolysis, more than IO substrates have been tried for their sensitivity against ChE in this experiment. These substrates included naphthyl esters, indoxyl esters and their substitute analogs, indophenyl acetate, etc. As judged by the rate of reactivity as well as the stability and precise location of
wzyme band ii1 t.he zyniograni. N h’l l,L\,was fonnci to l)e the best su1tstrat.e. A zymogram rc5ulLing from licrr~c~wr~m ChE t cr~v:ird t,his substrate i5 shown in Fig. 1. As cm 1~ well ii-0m this figures. tlw applied enzyme as low as 0.01 unit per t’ubc coul(.l bc detected. This i,+ especially useful for evaluating preparations at the low ChE level found in insects. 3loreover, the use of NMIA has several ad\x~tagos over otlier sulx$rates: (.I) The react’ion is very rapid. Clex c~~~zyrnc bandt; de\-eloped within a few minutes after the addit,ion of the incubatjing solut’ion when the applied enzyme was I unit per t’ube or more. (2) The colorless substrate is cnzymically hydrolyzed and converted into a greenish material (see II or III below) which cm be easily recognized by its int’ense color. (3) In the presence of oxygen, t,he enzymically hydrolyzed mat,erial I$‘-methyl indoxyl (IIj dimerized automatically and the final product is a highly insoluble greenish precipitate (,IV) (7) ; no coupling reagent such as diazonium, potassium cyanide, copper sulfide, etc. is required. The overall reaction is shown in Scheme I, As previously reported by Guilbault et aE. (7), NMIA is not specific,
3
1.
0.5
0.1
-
ChE Bands
-
Dye front
0.01 Units
of
ChE
FIG. 1. Electrophoretic patterns of horse serum ChE obtained by incubating with N-methyl indoxyl acetate. Enzyme concentrations range from 3 to 0.01 unit (units are expressed as pmoles acetylcholine chloride hydrolyzed per minute at 25°C and pH 7.4). Picture taken 5 minutes after incubation. Tucubation solution (6).
DETECTION
L N-Methyl Acetate
Indoxyt
IN
POLYACRYLAMIDE
GELS
11 N-Methyl Indoxyl Green
CWOLINE!STERASE
75
III N-Methyl Indoxyl Green
IV N-Methyl indigo Green SCHEME
I
but is hydrolyzed by lipase and cellulase, Substrate specificity towards both enzymes has been tested and our results indicated t)hat lipase had very low reactivity against this substrate in the gel. UsuaIly it took more than 12 hours to obtain clear enzyme bands. In this trial cellulase was almost inactive since no color band was observed, This may be due either to the inactivation of this enzyme during elect,rophoresis,or to the low reactivity of this enzyme toward the substrate. The activity of ChE from different sources toward this substrate has also been tested. In general, sharp enzyme bands of high color intensit’y were always obtained with insect ChE and widespread enzyme bands were observed with serum and liver ChE. Our previous studies on the molecular size estimation for these partially purified enzymes on SsephadexG-100 indicated that insect ChE appeared to be larger molecules than those of liver and serum ChE. The specific activity of the insect ChE used in this experiment was also lower compared with liver and serum CHE, It appears, therefore, that the larger insect ChE molecule might have slowed down the rapid diffusion of the enzyme in the gel after electrophoresis since the specificity of insect ChE used in this experiment was much lower t’han that of liver and serum ChE. EfJect of Incubatin-g System
Even though NMIA is much more soluble in systems containing organic solvents, only slight differences were obtained from systems with and without the addition of organic solvents. Generally speaking, the development of color enzyme bands was faster, and the precipitate formed in the gel was finer, in systems with organic solvents. This may be due to the
fact’ that tlw organis so1vcnt.s t~cill) tv bring more wbstrate ink) t,he incubating syst’em, and this high ;;ubstrate concc?nt,ration accelerated &he rate of penetration and reactioll. I-Iowevcr, the gradual development’ of a greenish background in noncnzylnc~ areas tended to obscure the exact localization of t’he enzyme hand. This interference became a serious problem when samples contained v(lry lmowenzyme activity. It is believed that the formation of the greenish background WRY clue to the nonenzymic hydrolysis of the substrate since a gradual appearance of a light. g,reenish color was also found after the expo.Gurc of blank gels, without any addition of ChE, in the incubating solutions for the samt’ period of t’ime. This same type of behavior was observed in homogeneous solution (7). The effect of different buffers was not st’riking. Good results could be obtained with both buffers, but a IiMe greenish background could also be found on long standing. For the detectZion of preparations containing extremely low activity, System 7 is recommrnded even though much longer incubating time is required. Distinctj enzyme bancis with clear background were a.lways obtained in t,his system after a 10 hour incubation. Effect of Substrnte and Freshness of Medin Different lots of substrate were found to give different results, e.g., the newer the substrate the faster the reaction. It, was also found that the fresher the incubating media, the more rapid is the reaction taking place in the gel. Thus, to obtain best. results, the time hctween media preparation and the start of incubation should be kept minimum.
When the volume ratio of the gel freshly removed from the electrophoresis tube to the incubating solution was lj the enzyme band showed up very slowly and the band int.ensity was faint. The rat’e of color development as well as the color int,ensity increased as the volume of the incubating solution increased. The slow development of color enzyme band was possibly due to the presence of small amounts ,of the substrate in the incubating solution and this limited amount of the substrate was insufficient for optimum reaction in the gel. In addition, the leakage of the enzyme out of gel might, have occurred during long incubation times since t.he condition was unfavorable for prompt enzyme rclaction. -4 faint color enzyme band was thus obtained. For best results, the volume of incubating solution should be at least 5 times that of the gel volume.
Enzyme bands were rapidly dccolorized when the g,el was stored in either acidic or alkaline saluCirrn, Prolmonged storage of gels in distilled
DETECTION
IN
POLYACRYLAMIDE
GELS
CHOLIKESTERASE
77
water also led to a decrease in the band intensity and the gel pattern ultimately faded. However, the rate of fading may be reduced by storage of gels in either Tris or phosphate buffer at 0.1 M, pH 7.0. No dramatic change in band intensily was noted within 20 days. SUMMARY
A convenient method for the rapid and precise dekction of cholinesterase (ChE) in polyacrylamide gels is described. A vastSly increased sensitivity was achieved by the use of N-methyl indoxyl acetat’e as substrate. As low as 1 x IO-” unit ChE can be detected within a few minutes by simply immersing the gel cylinder in the incubat,ing mixture. X:0 coupling reagent is required. ACKNOWLEDGMENT The financial Health Service, acknowledged.
supportof the Department NationaI
Institutes
of Health. Education and Welfare, Pub’i~ of Health, Grant ES 0042601, is gratefuli)
REFEREXCES 1. BARKA, T., AND ANDERSON, P. J. “Histochemistry, Theory, Practice and Bibliography,” p, 203. Hoeber (Harper & Row), New York, 1963. 2. BUR.STONE,M. S., J. N&l. Cancer Inst. 18, 167 (1957). 3. PEARSON,B., AND GROSE,F., A. M. A. Arch. Path&g. 67, 324 (1959). 4. SAVAY, GY., AND CSILLIK, B., Acta H&to&em. 6, 307 (1959). 5. GEREBTZOFF,M. A., Acta Anat. 19, 366 (1953). 6. ZWEIG, G., AND WHITAKER, J. R., “Paper Chromatography and Electrophoresis,” Vol. I, p. 176. Academic Press, New York. 1967. 7. GUILBAWLT, G. G., SADAR, M. H.. GLMER, R., AND SKOU. C.. Anal. Letters 1, 365 (1968). 8. GUILBAULT, G. G., SADAR,M. H., KAUN, S. S.. AND CASEY, D.. “The E&et of Pesticides on Liver Cholinesterases from Rabbit, Pigeon, Chicken, Sheep and Pig,” Anal, Chim. Acta, in press. 9. GUILB.~ULT, G. G., KUAN, S. S., AND SADAR,M. H., “Purification and Properties of Cholinesterases from Honey Bees-Apti millifera Linnaeus and Boll WeevilsAnthonamus grandk Boheman,” J. Agr. Food Chem., in press. 10. DAVIS, B. J., “Disc Electrophoresis,” Ann. N. Y. Acad. Sci. 121, 404 (1964).