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A simple, rapid, and quantitative radiometric assay of acetylcholinesterase
ANALYTICAL
BIOCHEMISTRY
A Simple,
57, 585-592
Rapid, Assay
Rewired
(1974)
and
Quantitative
Radiometric
of Acetylcholinesterase
June
11. 1973;
acceptrd
August
15. 1973
A thin-layer chromntographic method has been dewloped for the metric assay of minute quantities of acctylcholinesterase. It is simple, and allows the simultaneous assay and total recovcrirs of acetglcholinr ncetate.
radiorapid. and
There are many methods for assaying cholinesterase (ChE) activity such as bioassay, titrimetric, spectrophotometric, fluorometric, manometric, gas chromatographic, and radiomctric. Tht most scnsitire are the last three, of which only the radiometric is simple and fast. Radiometric assays have used substrates labeled in the acyl moiety, and followed either the radioactive acid (l-7) or the unhydrolyzed substrate (8 1, after separating them by volatilizat,ion (8). ultrafiltration (6 I, extraction and ccntrifugation ( l), or precipitation of unreactetl substrate by adsorption on cation exchange resin 14,71 or reaction with acidified ammonium reineckatc (2,3 1 followed by centrifugation. Except for the volatilization method (,8 1 these tcchniqucs are efficient in detecting minute quantit’ies of produced acid; the limit of their scnsitjivity is determined largely by the specific actirity of the substrate used. The elcctrophoresis mctliud (5’1 is rclat’ivcly slow, precipitation as rcincckatc salt is faster (2,3), and the others are extremely fast. Each of the radiomctric techniques has certain advantages such 3.5 high spcccl (6) and total acetate recorery 12). 1Iorcorer, spcetl. combined with low initial substrate concentration and minimal dilution can make the radiometric method relatiuclp sensitiw to choliwstwase inhibition by carbamates which behave as reversible inhibitors (9) Also, the ultrafiltration (6) and extraction ( 1) methods are qwcially adapted for the assay of micro~olumes. On the other hand, there are disarlrant~ages to the use of each tcchniqur. Removal of unh~tlrolyzctl substrate by adsorptiou on cation exchange resin (4 1 was judged as rc~latiwly inefficient ant1 not adaptable to microquantities (2), and c~xtraction of the acetate 1)~ toluene-isoamyl alcohol ( 1) was slion-n not to be quantitative (2). Copyright All rights
@ 1974 by Acadrmic Prrss, of rel~rotluction in any form
Inc. rrsrrvvd.
590
LEWIS
AND
ELDEFRAWl
ACh I
n
Acetate
I 123456
7
8
cm 0
F
FIG. 1. Histogram of a typical chromatogram of a mixture L”HIACh. 0. origin of spot on chromstogmm; P, front. Ination solution
of recoveries,
trip1icat.e
and their radioactivity
of [“HIacetate
and
aliquots were taken from the [“H]ACh counted.
When [“H]sodium acetate and [“H]ACh were spot’ted together and chromatographecl, the two compounds separated totally i Fig. 1 I. The Rf values, tletermined after checking each compound alone as well, were about 0.1 and 0.9 for the ACh and acetate, respectively. The same separation was obt,ained after enzyme hydrolysis of ] RH]ACh. Since acetate is easily lost by evaporation, recovery of [:‘H] sodium acetate was determined and found to be quantitative (Fig. 21, xv-henthe chromatogram was cut and put in scintillation cocktail right after development. Total recovery was also obtained for (“H]ACh. The recoveries of bot,h AC11 and acetate were reduced t.he longer the time interval was between the development of the chromatogrnm and put,ting the cut pieces into the cocktail. For exampfc, acetate recoveries were 100, 90, and 7CJs after 0, 10, and 30 min, respectively. Hydrolysis of [‘lH]ACh by AChE was calculated by dividing the radioactivity present as 13H]acetate by either the total radioactivity a&led to t)he chromatogram, or by the sum of radioactivity present as (“H]acctate and as ]“H]ACh on the chromatogram at the end of the run. There was a linear relationship between percent hydrolysis and the amount of AC’hE uacd t Fig. 3 1. Spontaneous hydrolysis during the 20 min run was calculated to he 1.356 at IO pM ACh, as the Y-intercept indirntcs. Hydrolysis by AChE was progressive and also linear up to the 25 lnin twted. Re-
ASSAY
OF
BCETTLCHOLINESTEHASE
591
E’K. 2. Rcrowr~of [“HIacetate from 1.11~ c*hromatogram. The absrissa is for 5 pl smn~~lcs from stock solutions. whrrcns the ortlinnle is the total cpm recovered from thcx cllromntopram derc-loped after the addition of 5 pl of tlrc* same soluiions of acct,:rt,c. Thcl chromntogmm was cut immediately after development.
I:Ic;.
cuhxtion bars arr
3. l’-‘c,r.ct>nl Ilydt olyris time was 20 min mcnns and stnndnrd
of L”HIACh by incrrnsing and [“HI AC% conccntrxtion errors of six experiments.
concentrat,ions of AChE. In10 pn1. Dots and rrrticnl
592 1)roducibility terminations
LEWIS
was q&e was 2.3%.
AND
ELDEFRAWI
good, and the standard
deviation
of three de-
DISCUSSION
The thin-layer chromatography method for the assay of AChE activity is simple, rapid, and adapt’able to the simultaneous analysis of many samples. It is efficiwt in totally separating t.lw acetate from XC’11 (Fig. 1) and is quantitative (Fig. 2), qualities which mnke it superior to the organic extraction method (1). The ultrafiltration method (6 I may be as fast, but recoveries were not given and there may be loss of acetate by evaporation. The present method is equal in sensitivity to the extraction and reineckate precipitation methods 11,2), but has the advantage over the latter, and even its microadaptation (3), by its ability to detect smaller quant,ities. These three techniques seem to be equally sensitive, the limit of their sensitivity being the specific activity of the [“H]hCh used. In the present approach, ,4ChE act’ivity is terminat.ed by the chromatographic solvent (80% ethanol) . The other methods have used mainly llydrocllloric acid (l-5,7), which may by itself cause further hydrolysis of ACl1. Ncostigmine was used in conjunction with tl1e ultrafiltration method (6)) but since carbamylation of the enzyme is progressirc, AChE may continue bring act,ire for a few more minutes. In conclusion, we feel tllat, our technique combines the advantages of encl1 of the other racliomctric ones arid also permits the simultaneous assay not, only of acetate but. also of tl1e unreacted ACh. Thin-layer chromatography 11~ been used to separate choline from AC11 (11’~. and the method should also bc effective for other clloline esters.
1. POTTER, L. 2. MCCAMAS, 3. Ii.
5. 6. 7. S.
9. 10. 11.
T.
(1967)
M.
UT.,
J. Phrt~maco2. Errp. l’her. 156, 500. TOMEY, I,. H.. AND MCCA;MAN, R. E. (1968) Life Sci. 7, 233. K~~LIJ~, S. H.. AND GIACOBINI, E. (1969) J. Neurochem. 16, 1523. REED, D. J., GOTO, Ii., AND WANG, C. H. (1966) Ann/. Riochem. 16, 59. POTTER. I,. T.. .ASD M~~RI’IIT. W. (1967) Biochem. Phat-macol. 16, 1386. EHRENPREIS. S., MITT.K> T. W., AND PATRICK. P. (1970) Biochem. Pharmacol. 19, 2165. hADY, c. 11.. AND I
BIOCHEMISTRY
A Simple,
57, 585-592
Rapid, Assay
Rewired
(1974)
and
Quantitative
Radiometric
of Acetylcholinesterase
June
11. 1973;
acceptrd
August
15. 1973
A thin-layer chromntographic method has been dewloped for the metric assay of minute quantities of acctylcholinesterase. It is simple, and allows the simultaneous assay and total recovcrirs of acetglcholinr ncetate.
radiorapid. and
There are many methods for assaying cholinesterase (ChE) activity such as bioassay, titrimetric, spectrophotometric, fluorometric, manometric, gas chromatographic, and radiomctric. Tht most scnsitire are the last three, of which only the radiometric is simple and fast. Radiometric assays have used substrates labeled in the acyl moiety, and followed either the radioactive acid (l-7) or the unhydrolyzed substrate (8 1, after separating them by volatilizat,ion (8). ultrafiltration (6 I, extraction and ccntrifugation ( l), or precipitation of unreactetl substrate by adsorption on cation exchange resin 14,71 or reaction with acidified ammonium reineckatc (2,3 1 followed by centrifugation. Except for the volatilization method (,8 1 these tcchniqucs are efficient in detecting minute quantit’ies of produced acid; the limit of their scnsitjivity is determined largely by the specific actirity of the substrate used. The elcctrophoresis mctliud (5’1 is rclat’ivcly slow, precipitation as rcincckatc salt is faster (2,3), and the others are extremely fast. Each of the radiomctric techniques has certain advantages such 3.5 high spcccl (6) and total acetate recorery 12). 1Iorcorer, spcetl. combined with low initial substrate concentration and minimal dilution can make the radiometric method relatiuclp sensitiw to choliwstwase inhibition by carbamates which behave as reversible inhibitors (9) Also, the ultrafiltration (6) and extraction ( 1) methods are qwcially adapted for the assay of micro~olumes. On the other hand, there are disarlrant~ages to the use of each tcchniqur. Removal of unh~tlrolyzctl substrate by adsorptiou on cation exchange resin (4 1 was judged as rc~latiwly inefficient ant1 not adaptable to microquantities (2), and c~xtraction of the acetate 1)~ toluene-isoamyl alcohol ( 1) was slion-n not to be quantitative (2). Copyright All rights
@ 1974 by Acadrmic Prrss, of rel~rotluction in any form
Inc. rrsrrvvd.
590
LEWIS
AND
ELDEFRAWl
ACh I
n
Acetate
I 123456
7
8
cm 0
F
FIG. 1. Histogram of a typical chromatogram of a mixture L”HIACh. 0. origin of spot on chromstogmm; P, front. Ination solution
of recoveries,
trip1icat.e
and their radioactivity
of [“HIacetate
and
aliquots were taken from the [“H]ACh counted.
When [“H]sodium acetate and [“H]ACh were spot’ted together and chromatographecl, the two compounds separated totally i Fig. 1 I. The Rf values, tletermined after checking each compound alone as well, were about 0.1 and 0.9 for the ACh and acetate, respectively. The same separation was obt,ained after enzyme hydrolysis of ] RH]ACh. Since acetate is easily lost by evaporation, recovery of [:‘H] sodium acetate was determined and found to be quantitative (Fig. 21, xv-henthe chromatogram was cut and put in scintillation cocktail right after development. Total recovery was also obtained for (“H]ACh. The recoveries of bot,h AC11 and acetate were reduced t.he longer the time interval was between the development of the chromatogrnm and put,ting the cut pieces into the cocktail. For exampfc, acetate recoveries were 100, 90, and 7CJs after 0, 10, and 30 min, respectively. Hydrolysis of [‘lH]ACh by AChE was calculated by dividing the radioactivity present as 13H]acetate by either the total radioactivity a&led to t)he chromatogram, or by the sum of radioactivity present as (“H]acctate and as ]“H]ACh on the chromatogram at the end of the run. There was a linear relationship between percent hydrolysis and the amount of AC’hE uacd t Fig. 3 1. Spontaneous hydrolysis during the 20 min run was calculated to he 1.356 at IO pM ACh, as the Y-intercept indirntcs. Hydrolysis by AChE was progressive and also linear up to the 25 lnin twted. Re-
ASSAY
OF
BCETTLCHOLINESTEHASE
591
E’K. 2. Rcrowr~of [“HIacetate from 1.11~ c*hromatogram. The absrissa is for 5 pl smn~~lcs from stock solutions. whrrcns the ortlinnle is the total cpm recovered from thcx cllromntopram derc-loped after the addition of 5 pl of tlrc* same soluiions of acct,:rt,c. Thcl chromntogmm was cut immediately after development.
I:Ic;.
cuhxtion bars arr
3. l’-‘c,r.ct>nl Ilydt olyris time was 20 min mcnns and stnndnrd
of L”HIACh by incrrnsing and [“HI AC% conccntrxtion errors of six experiments.
concentrat,ions of AChE. In10 pn1. Dots and rrrticnl
592 1)roducibility terminations
LEWIS
was q&e was 2.3%.
AND
ELDEFRAWI
good, and the standard
deviation
of three de-
DISCUSSION
The thin-layer chromatography method for the assay of AChE activity is simple, rapid, and adapt’able to the simultaneous analysis of many samples. It is efficiwt in totally separating t.lw acetate from XC’11 (Fig. 1) and is quantitative (Fig. 2), qualities which mnke it superior to the organic extraction method (1). The ultrafiltration method (6 I may be as fast, but recoveries were not given and there may be loss of acetate by evaporation. The present method is equal in sensitivity to the extraction and reineckate precipitation methods 11,2), but has the advantage over the latter, and even its microadaptation (3), by its ability to detect smaller quant,ities. These three techniques seem to be equally sensitive, the limit of their sensitivity being the specific activity of the [“H]hCh used. In the present approach, ,4ChE act’ivity is terminat.ed by the chromatographic solvent (80% ethanol) . The other methods have used mainly llydrocllloric acid (l-5,7), which may by itself cause further hydrolysis of ACl1. Ncostigmine was used in conjunction with tl1e ultrafiltration method (6)) but since carbamylation of the enzyme is progressirc, AChE may continue bring act,ire for a few more minutes. In conclusion, we feel tllat, our technique combines the advantages of encl1 of the other racliomctric ones arid also permits the simultaneous assay not, only of acetate but. also of tl1e unreacted ACh. Thin-layer chromatography 11~ been used to separate choline from AC11 (11’~. and the method should also bc effective for other clloline esters.
1. POTTER, L. 2. MCCAMAS, 3. Ii.
5. 6. 7. S.
9. 10. 11.
T.
(1967)
M.
UT.,
J. Phrt~maco2. Errp. l’her. 156, 500. TOMEY, I,. H.. AND MCCA;MAN, R. E. (1968) Life Sci. 7, 233. K~~LIJ~, S. H.. AND GIACOBINI, E. (1969) J. Neurochem. 16, 1523. REED, D. J., GOTO, Ii., AND WANG, C. H. (1966) Ann/. Riochem. 16, 59. POTTER. I,. T.. .ASD M~~RI’IIT. W. (1967) Biochem. Phat-macol. 16, 1386. EHRENPREIS. S., MITT.K> T. W., AND PATRICK. P. (1970) Biochem. Pharmacol. 19, 2165. hADY, c. 11.. AND I