Preparative Biochemistry and Biotechnology

ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20

Purification and Characterization of

Acetylcholinesterase from the Lake Van Fish
(Chalcalburnus tarichii Pallas, 1811)

Serpil Aliriz & Vedat Turkoglu

To cite this article: Serpil Aliriz & Vedat Turkoglu (2003) Purification and Characterization of
Acetylcholinesterase from the Lake Van Fish (Chalcalburnus�tarichii Pallas, 1811), Preparative
Biochemistry and Biotechnology, 33:2, 137-145, DOI: 10.1081/PB-120021438

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 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
Vol. 33, No. 2, pp. 137–145, 2003

Purification and Characterization of

Acetylcholinesterase from the
Lake Van Fish
(Chalcalburnus tarichii Pallas, 1811)

Serpil Aliriz and Vedat Turkoglu*

Science and Arts Faculty, Department of Chemistry,

Yuzuncu Yil University, Van, Turkey

ABSTRACT

In this study, acetylcholinesterase (AChE; EC 3.1.1.7) was purified

from plasma and erythrocytes in the Lake Van fish (Chalcalburnus
tarichii P.1811) by affinity chromatography. Enzymatic activity was
spectrophotometrically measured according to Ellman’s method,
at 412 nm. Then, the optimal pH and temperature of the enzyme
was determined. According to the results, the optimal pH and the
optimum temperature were 8.0 and 25
C, respectively. In order to
control the purification of the enzyme, sodium dodecyl-
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done.

*Correspondence: Vedat Turkoglu, Science and Arts Faculty, Department of

Chemistry, Yuzuncu Yil University, Van, Turkey; E-mail: vturkoglu_62@
yahoo.com.

137

DOI: 10.1081/PB-120021438 1082-6068 (Print); 1532-2297 (Online)

Copyright # 2003 by Marcel Dekker, Inc. www.dekker.com
138 Aliriz and Turkoglu

SDS-PAGE showed a single band for enzyme. The purification rates

for plasma AChE and erythrocyte AChE are 3251.6 and 8500,
respectively.

Key Words: Purification; Acetylcholinesterase; Affinity chromato-

graphy; Van Lake Fish (Chalcalburnus tarichii P.1811).

INTRODUCTION

Acetylcholinesterase (EC 3.1.1.7) catalyses the hydrolysis of acethyl-

choline, a neurotransmitter substance which functions in certain portions
of the nervous system. The reaction catalysed by acetylcholinesterase
occurs enzymically in two steps. In the first step, the enzyme serves as
a remarkable nucleophile,
O
k
ðCH3 Þ3 Nþ C2 H4 O-C-CH3 þH2 O$ðCH3 Þ3 Nþ C2 H4 OHþCH3 COOH
and, in the second step, the enzyme serves as an excellent leaving group.
The nucleophilic group is the hydroxyl group of a specific serine residue.[1]
O O
k k
ðCH3 Þ3 Nþ C2 H4 O-C-CH3 þE:$CH3 -C -EþðCH3 Þ3 Nþ C2 H4 OH
$

CH3 COOHþE
Later, such enzymes were found in blood. Both serum and red cells
hydrolyse choline esters far more rapidly than other esters at low subst-
rate concentrations. The red cell type has been called acetylcholinesterase.
Acetylcholinesterase is found in all conducting tissue in all species of ani-
mals which have been investigated. Acetylcholinesterase is mainly found
in the brain, in nerve cells (especially end plates), in muscle, and in eryth-
rocytes. Acetylcholinesterase was first isolated by extraction from the
electric organ of Torpedo marmorata in 1938, following the discovery of
an extraordinary concentration of the enzyme in this tissue.[2] The pH
optimum of the AChE of plasma and erythrocytes is around pH 8.0. Both
enzymes have a distinctly different optimum substrate concentration.[3]
The aim of this study is to purify acetycholinesterase enzyme in
Chalcalburnus tarichii, a fish species living in Lake Van (the pH is 9.5),
which has a maximum depth of 450 m, by affinity chromatography, and
to characterise and to compare purified enzyme from Chalcalburnus
tarichii with those of the other living species.
Acetylcholinesterase in Lake Van Fish 139

EXPERIMENTAL

Materials

The chemicals used in the present study are: Sepharose 4B, EDCl (1-
ethyl-3-(3-dimethyl-aminopropyl) carbadiimide hydrochloride), Sepha-
dex G-25, decamethonium bromide, standard bovine serum albumin,
Coomassie brillant blue G-250, Coomassie brillant blue R-250,
N,N,N0 ,N0 -tetramethylethylene diamine (TEMED), trichloroacetic acid
(TCA), sodium dodesilsulfate (SDS), dialysis bag, and NaOH were pur-
chased from Sigma Chem. Co. Sodium carbonate, sodium bicarbonate,
trihydroxymethyl aminomethane (Tris), NaCl, cyanide bromide (CNBr),
sodium citratedehydrate, HCl, acetic acid, b-mercaptoethanole, propa-
nol, and methanol were purchased from Merck. The blood samples with
ACD (acid-citrate-dextrose) were obtained from Chalcalburnus tarichii.

Methods

Preparation of the Hemolysate

The blood samples were obtained by using bottles with anticoagulant

(ACD) from Chalcalburnus tarichii. The blood samples were centrifuged
at 1500 r.p.m for 20 min and the plasma and buffy coat were removed.
After the packed red cells were washed with NaCl (0.9%) three times,
the erythrocytes were hemolyzed with cold water. The membrane and
intact cells were centrifuged at 12,000 r.p.m for 30 min and the superna-
tant was removed. This process was repeated three times and a tempera-
ture of 4
C was maintained during the process. The pH of plasma and
erythrocytes were adjusted to 8.0 by adding sodium phosphate (25 mM)
and solid phosphate salts.

Preparation of Affinity Gel

After 20 mL of Sepharose 4B and 20 mL of water were combined,

4 g of CNBr was added to this suspension. The mixture was titrated
to pH 11 in an ice bath by stirring with a magnet, and maintained at
that pH for 8–10 min. The reaction was stopped by filtering the gel
onto a Buchner funnel and washing with cold 0.1 M NaHCO3 buffer
(pH 10). 1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochlor-
ide, using saturated EDCl solution in the same buffer, was coupled to
Sepharose 4B-EDCl activated with CNBr. The reaction was completed
by stirring with a magnet for 90 min. In order to remove excess of
140 Aliriz and Turkoglu

1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride from

the Sepharose 4B-EDCl gel, the mixture was washed with an abundance
of water. The affinity gel was suspended in buffer A (50 mM sodium
phosphate, pH 8) then packed into a column (1.3  60 cm) and equili-
brated with the same buffer. The flow rates for washing and equilibra-
tion were adjusted by peristaltic pump at 40 mL=h.

Purification of AChE by Affinity Chromatography

The hemolysate was applied to the affinity column. Hemolysate

sample obtained previously was loaded onto a Sepharose 4B-EDCl affinity
column and the flow rate was adjusted to 20 mL=h. The column was
washed with buffer B (0.4 NaCl, 50 mM sodium phosphate pH 8) until
the A280 nm of the effluent was  0.01. The AChE was eluted with buffer
C (0.1 M NaCl, 50 mM sodium phosphate pH 8, containing 10 mM deca-
methonium). The enzyme activity was measured in the final fractions and
the activity-containing tubes were collected together. Then the enzyme
solution was dialysed in buffer A for 3–4 h with two changes of buffer.[4–8]

Activity Determination

For the colorimetric assay, according to Ellman’s method, reaction

mixtures were made up in 50 mM Tris, pH 7.4, containing 1 mM DTNB
and acetylthiocholine iodide at a final concentration of 0.8 mM. The reac-
tion was performed at 25
C and monitored at 412 nm.[9]

Protein Determination

Quantitative protein determination was spectrophotometrically per-

formed at 595 nm according to Bradford’s method.[10]

Optimal pH Determination

For the optimal pH determination, the enzyme activity was measured

in 1 M Tris-HCl and phosphate buffers over the pH range of 6.0 to 11.0
and the optimal pH was found to be 8.0 for both plasma and erythrocytes.

Effect of Temperature on AChE Activity

The enzyme activity was measured between 10 and 60
C at optimal

pH for this purpose and the optimal temperature was found as 25
C
for both plasma and erythrocytes.
Acetylcholinesterase in Lake Van Fish 141

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Laemmli’s procedure was carried out at 4% and 10% acrylamide con-

centrations for running and stacking gel, respectively, to control enzyme
purity SDS added into the gel solution at 10%. The gel was stabilized in a
solution containing 50% propanol þ 10% TCA þ 40% distilled water for
30 min. The staining was done for about 2 h in a solution of 0.1% Coo-
massie Brillant Blue R-250 þ 50% methanol þ 10% acetic acid. Finally,
washing was carried out in a solution of 50% methanol þ 10% acetic
acid þ 40% distilled water until protein bands were cleared.[11]

RESULTS AND DISCUSSION

In this study, acetylcholinesterase was first purified from Chalcalbur-

nus tarichii. The purification steps included preparation of hemolysate
and Sepharose 4B-EDCl affinity chromatography. Table 1 shows a pur-
ification of plasma characterized with a spesific activity of 780.4 EU=mg
protein, a yield of 100%, and a purification coefficient of 3251.6. Table 2
shows a purification of erythrocyte characterized with a specific activty of
23.80 EU=mg protein, a yield of 71%, and a purification coefficient of
8500. Sepharose 4B was chosen as a matrix because of its better flow
properties compared with other matrices. At first, free hydroxyl groups

Table 1. Purification of plasma AChE from Chalcalburnus tarichii.

Total Total Total Specific

Purification volume protein activity activity Yield Purification
steps (ml) (mg) (EU) (EU=mg) (%) factor

Hemolysate 150 10020 16.1 0.24 100 0

Affinity column 50 1.03 16.1 780.4 100 3251.6

Table 2. Purification of Erythrocyte AChE from Chalcalburnus tarichii.

Total Total Total Specific

Purification volume protein activity activity Yield Purification
steps (ml) (mg) (EU) (EU=mg) (%) factor

Hemolysate 150 7545 14.1 0.28 100 0

Affinity column 80 0.337 10.02 23.80 71 8500
142 Aliriz and Turkoglu

Figure 1. Purification of plasma AChE by affinity chromatography. The columns

(1.3  60 cm) eluted by buffer C at pH 8. Buffer at 20 mL=hr flow rate for fraction
volumes of 6 mL.

of the matrix were activated with cynogen bromide.[12] The AChE from
erythrocytes and plasma were purified by using the affinity gel with elu-
tion buffer (0.1 M NaCl, 10 mM sodium phosphate pH 8.0 containing
10 mM decamethonium). The eluates were plotted by carrying out pro-
tein determination at 280 nm and AChE activity for plasma (Fig. 1)
and erythrocytes (Fig. 2). Specific activities for plasma and erythrocytes
were calculated by using hemolysate and purified enzyme solutions.

Figure 2. Purification of erythrocyte AChE by affinity chromatography. The

column (1.3  60 cm) was eluted by buffer, pH 8. Buffer for AChE at 20 mL=h
flow rate for fraction volumes of 6 mL.
Acetylcholinesterase in Lake Van Fish 143

Optimal pH of AChE for erythrocyte and plasma has been determined

as 8.0, using 1 M Tris- HCl and are shown in Fig. 3 and Fig. 4, respectively.
The pH determined was similar to that in the previous studies.[13,14] The
enzyme activities of plasma and erythrocyte were measured at 25
C and
are shown in Fig. 5 and Fig. 6, respectively.

Figure 3. Activity-pH plot of erythrocyte AChE.

Figure 4. Activity-pH plot of plasma AChE.

Figure 5. The effect of temperature on plasma AChE.

144 Aliriz and Turkoglu

Figure 6. The effect of temperature on erythrocyte AChE.

Figure 7. SDS-polyacrylamide gel electrophoresis of AChE purified by affinity

chromatography.

There was a 3251.6-fold purification of plasma AChE and an 8500-

fold purification of erythrocyte AChE. The purification of AChE was
controlled with SDS-polyacrylamide gel electrophoresis (Fig. 7).
So far, no study examining this species of fish has been done for the
enzyme. Because of this, we did not have a chance to compare our results
with previous results. In addition, because of high variability in analyz-
ing enzyme in vitro and in vivo, due to inconsistent factors such as treat-
ment time and manner, purity and species tissue differences, etc., it is
difficult to compare data from different laboratories regarding the toxico-
logical effects.
Acetylcholinesterase in Lake Van Fish 145

REFERENCES

1. Lehninger, A.L. Principles of Biochemistry; Worth Publisher, Inc.:

New York, 1982; 222–223.
2. Nachmansohn, D.; Lededer, E. Chemical and molecular basis of
nerve activity. Bull. Soc. Chem. Biol. 1939, 21, 797.
3. Pilz, W. Klin. Wschr. 1958, 36, 1017.
4. Brewer, G.J.; Sing, C.F. An Introduction to Izoenzymes Techniques;
Academic Press, Inc.: New York, 1970; 207.
5. Hoz, D.L.D.; Raltson, J.S.; Rush, R.S.; Wolfe, A.D. A simplified
procedure for the purification of large quantities of fetal bovine
serum acetylcholinesterase. Life Sci. 1986, 39, 195–199.
6. Raltston, J.S.; Rush, R.S.; Doctor, P.B.; Wolfe, A.D. Acetylcholin-
esterase from fetal bovine serum purification and characterisation of
soluble G4 enzyme. J. Biol. Chem. 1985, 260 (7), 4312–4318.
7. Main, A.R.; Soucie, W.G.; Buxton, L.I.; Arinc, E. The purification
of cholinesterase from horse serum. Biochem. J. 1974, 143, 733–744.
8. Bruns, K.; Casillas, E.D. Partial purification and characterization of
an acetylcarnitine hydrolase from bovine epididymal spermatozoa.
Arch. Biochem. Biophys. 1990, 277 (1), 1–7.
9. Ellman, G.L.; Courtney, K.D.; Andres, V. Jr.; Featherstone, R.M.
A new and rapid colorimetric determination of acetylcholinesterase
activity. Biochem. Pharmacol. 1961, 7, 88–95.
10. Bradford, M.M. A rapid and sensitive method for the quantitation
of microgram quantaties of protein utilizing the principle of protein-
dye binding. Anal. Biochem. 1976, 72, 248.
11. Laemmli, N.K. Cleavage of structural proteins during in assembly
of the heat of bacteropose T4. Nature 1970, 227, 680.
12. Bell, J.E.; Bell, E.T. Affinity Chromatography. Protein and Enzymes;
Prentice-Hall, Inc.: New Jersey, 1988; 45–63.
13. Ulusu, N.N.; Kus, M.S.; Acan, L.; Tezcan, E.F. Intl. J. Biochem.
Cell Biol. 1999, 31, 787.
14. Bilgi, C.; Unsal, I.; Ozer, N.; Erbil, M.K.; Karaca, L. Tr. J. Med.
Sci. 1995, 27, 291.

Received October 28, 2002

Accepted December 3, 2002
Manuscript 7251