Eur. J. Biochem. 96, 151-158 (1979)
Cobra Venom Acetylcholinesterase Purification and Molecular Properties Raivo RABA. Aavo AAVIKSAAR, Mari RABA, and Juri SIlGUR Biochemistry Department, Institute of Cybernetics of the Academy of Sciences of the Estonian SSR, Tallinn (Received August 14. 1978iJanuary 4, 1979)
Acetylcholinesterase from cobra (Nu@ naja oxiunu) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67000 i-2000. At high enzyme concentrations ( > 0.2 mg/ml, > 1 pM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1 -S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.
In recent years several acetylcholinesterases from animal tissues (electric organ of fishes, erythrocytes and brain of mammals etc.) have been purified up to a high degree of purity [I - 71. This has led to intensive work on the molecular structure of the enzyme. The investigations have elucidated a remarkable heterogeneity of acetylcholinesterases from various sources. In the case of electroplax there exist at least six forms of different size and shape [S]. Analogous multiple forms have been obtained for the erythrocyte enzyme [9,10]. In the detergent-free medium mammalian acetylcholinesterase also irreversibly forms aggregates of different molecular weights [ll]. In addition, as has been confirmed by Brodbeck et al. [12], any of these multiple forms of electric organ and erythrocyte acetylcholinesterase can be separated further into several fractions by isoelectric focusing. Although the catalytic specificity of Elapidae venom acetylcholinesterases has been shown to coincide with the specificity of the erythrocyte enzyme [13- 161, the venom enzymes seem to have a much less complicated structure than the acetylcholinesterase from membranes. Kumar and Elliott [5] who have investigated Bungurus,fizsciutus venom acetylG i : j . , i i e . s . Acetylcholinesterase o r acetylcholine acetyl-hydrolase (EC 3.1.1.7): ncuraminidase (EC 3.2.1.18). R’c~tc.This is the third paper in a series on C O ~ TVCPKJVI LI ACCIVIc./io/i~7r,.sir,.trsc.:the second paper appeared elsewhere [14].
cholinesterase, found only a single dimeric form of the enzyme with the molecular weight of 126000, consisting of identical catalytically active subunits. In a more recent work Lee, Latta and Elliott [17] reported that they have found from three elapid venoms several acetylcholinesterase isoenzymes differing in charge. Considering the similarities in catalytic properties of venom and tissue acetylcholinesterases we assume that the venom enzyme could serve as a convenient model for studying acetylcholinesterase mechanisms. In this work we describe the purification of cobra (Nuju nuju oxiunu) venom acetylcholinesterase. Molecular properties of the enzyme have been investigated and compared with other acetylcholinesterases.
MATERIALS AND METHODS Muteriuls
Desiccated Middle-Asian cobra (Nuju naju o.xiunu) venom was purchased from the Taschkent Integrated Zoo Plant (Uzbek SSR). Marker proteins used were : ovalbumin, catalase, chymotrypsinogen (all from Reanal, Hungary), cytochrome c (Serva, Heidelberg), cc-chymotrypsin (Worthington) and bovine serum albumin (Koch-Light laboratories, Colnbrook).
152
Cobra Venom Acetylcholinesterase
Neuraminidase (Vibrio cholerae, grade B, 500 U/ mg) was obtained from Calbiochem. Ampholine polyacrylamide gel plates pH 3.5- 10, were supplied by LKB Producter AB (Bromma). AH-Sepharose4B, Sephadex G-50 f, Sephadex G-100 sf and blue dextran 2000 were products of Pharmacia Fine Chemicals (Uppsala). Coomassie brilliant blue R250 ((2.1. 42660) was obtained from Ferak, fast blue RR salt from Reanal (Hungary) and basic fuchsin ((2.1. 42500) from Reachim (USSR). All other reagents used were of analytical grade.
Synthesis of Aflinity Adsorbent
N , N , N - Trimethyl - (m- aminophenyl) -ammonium chloride was synthesized according to Traylor and Singer [18]. This compound was quite a strong inhibitor for cobra venom acetylcholinesterase (Ki = 0.1 mM at 25 "C, pH 7.5, acetylcholine as substrate). Succinylated AH-Sepharose was prepared as described by Cuatrecasas and inhibitor was attached to the arm by carbodiimide coupling [19]. The affinity adsorbent had the following structure Sepharose 4B NH
-
(CH2)6- N H -CO
-
Assay of' Acetylcholinesterase Activity In the course of purification, acetylcholinesterase activity was determined spectrophotometrically with acetylthiocholine in the presence of a chromogenic agent 5,5'-dithio-bis(2-nitrobenzoic)acid as described by Ellmann et al. [20]. The reaction was carried out at room temperature on Specord UV-VIS spectrophotometer (Carl Zeiss, Jena). Acetylcholinesterase activity determination with acetylcholine was performed with the pH-stat method on a Radiometer TTT2/SBR3/ABU12 Titrigraph set. The reaction mixture contained 0.1 M NaCl, 0.01 M MgC12 and 1 mM acetylcholine. Acetylcholine hydrolysis was carried out at 25 "C and pH 7.5 under a stream of (202-free air. Activity of the enzyme was expressed in the international activity units (IU, pmol min- '). Activity localization in electrophoresis and isoelectric focusing experiments was performed by two different methods. The sensitive, although less specific, method of esterase activity staining with a-naphthyl acetate using the azo-coupling reagent fast blue RR salt was usually preferred. As established by Mounter [13], cobra venom does not contain any other enzyme
(CH2)2 - CO - NH -
Enzyme Purification All purification procedures were carried out at 4°C. Step I : Gel Filtration. 20 g of crude venom was dissolved in 0.2 M ammonium acetate pH 6.8, centrifuged to remove insoluble contaminants and passed through a column (110 x 11 cm) of Sephadex G-50 fine. Fractions containing acetylcholinesterase activity were pooled together and lyophilized. Lyophilized protein was stored at 4°C until use (up to 6 months). Step I I : Affinity Chromatography. 20 ml of affinity gel prepared as described was packed into a 14 x 1.4-cm column and equilibrated with 0.1 M sodium bicarbonate buffer pH 7.9, containing 0.2 M NaCl and 0.01 M MgC12. 100- 200 mg of acetylcholinesterase preparation from step I was dissolved in the elution buffer and dialyzed overnight. The sample was applied into the column and unadsorbed material was eluted with the buffer. For acetylcholinesterase elution, 0.05 M phenyltrimethylammonium iodide solution in the elution buffer was used. Eluted acetylcholinesterase was collected, concentrated by dialysis against 10 '%; poly(ethy1ene glycol) solution in 0.2 M NaCl, and finally dialysed against desired medium. Enzyme was usually stored in 0.1 M phosphate buffer pH 7.5, 0.2 M NaCI, at 4"C, or at -30°C for periods longer than one month. N o detectable loss of activity was observed under these conditions.
(0) Y N(CH3)3. +
capable of hydrolysing carboxylate esters. The gel was incubated after electrophoresis with 0.2 M phosphate buffer pH 7.0, for 5 - 10 min, then 4 ml of 0.5% a-naphthyl acetate and 2.5% fast blue RR salt solution in 50% acetone was added per 100 ml of the buffer solution, and mixed quickly. The reaction mixture was incubated for 5-30 min, depending on enzyme concentration. Alternatively, acetylcholinesterase was developed with acetylthiocholine according to Tripathi and O'Brien [7]. This method is specific and highly sensitive but the opaque enzyme bands obtained were less stable and produced poor photographic images. Protein concentration was determined by the modified Lowry method according to Hartree [21] using bovine serum albumin as standard.
Sedimentation Analysis The analysis was performed on Beckman Spinco E ultracentrifuge equipped with schlieren optics. The acetylcholinesterase sample was run at 60000 rev./min and 21 "C in 0.16 M NaCl at two different enzyme concentrations (2.8 and 1.4 mgiml). The values of were calculated in both cases. s20.
EIectroplzorc.sis Sodium dodecylsulfate/polyacrylamide gel electrophoresis was carried out in a discontinuous buffer
153
R. Raba, A. Aaviksaar, M. Raba, and J. Siigur Table 1 Purification of Naja naja oxiana venom acetylcholinrsteruse Step I1 was carried out on 200 mg of material from step I; the results obtained have been adjusted proportionately Total protein
Fraction
Total activity
Specific activity
Purification ~
~
~
~
.
overall
Crude venom I Gel filtration I1 Affinity chromatography
mg
IU
IU/mg
-fold
20000 2 635 34
263000 208 000 173900
13 1 78 8 5000
6 380
system according to Laemmli [22]. The gel tubes were stained immediately after electrophoresis with 0.1 Coomassie brilliant blue R250 solution in water/ ethano1:acetic acid mixture (7/7/1) for 2 h, destained with 7 (’( acetic acid in 25 ethanol and stored in 7 acetic acid. Disc electrophoresis was performed according to Ornstein and Davis in 7.5% gel at pH 8.3 [23]. The gel tubes were stained and destained as described by Chrambach et al. [24]. For glycoprotein staining, the periodic acid/Schiff reagent procedure described by Anselstetter and Horstmann was used 1251.
x
x
Gel Filtration For molecular weight determination, Sephadex G-100sf gel filtration media were used. The gel was packed into a column of 1.2 x 65 cm; 25 mM phosphate buffer solution pH 7.0 containing 0.1 M NaCl and 0.02”(; NaN3 was used for elution at a flow rate of 2.5 ml/h. Bovine serum albumin, ovalbumin and chymotrypsinogen were used as standard proteins. All the proteins were separately applied in 1-ml volume at 1 -2-mg/ml concentration into the column, and the column eluate was monitored by LKB Uvicord 111 analyzer at 281 nm to determine elution volume V,. Blue dextran 2000 was used as a void volume (VO) marker. A log M , versus Ve/Vo plot was used as a calibration curve in the molecular weight determination of acetylcholinesterase.
Isoelectric Focusing Analytical electrofocusing experiments were performed with LKB Multiphor apparatus using standard polyacrylamide gel plates, pH 3.5- 10. The plates were fixed and stained for protein as recommended by the manufacturer, LKB Producter AB (Bromma). Staining of the gel for enzyme activity was performed as described above. The pH on the plate surface was measured by means of a calibrated antimony/calomel electrode pair [26].
Recovery .~
.~ .~ .
..
. .
.
over preceding step
overall
over preceding step
6 63 5
79 66
19 84
Sialic Acid Determination and Neuraminidase Treatment Sialic acid content in the acetylcholinesterase molecule was determined according to Warren [27]. N-Acetylneuraminic acid was used as a standard. Neuraminidase treatment was carried out with Vibrio cholerae neuraminidase. To 0.4mlO. 1 M sodium acetate buffer (pH 5.5, 0.2 M NaCl, 0.02 M CaCb), 0.1 ml acetylcholinesterase solution (2 mg/ml, 0.2 M NaCl) and 0.1 ml neuraminidase solution (500 Ujml) were added. The mixture was incubated for 24 h or 96 h at 30 “C. Control experiments with human serum instead of acetylcholinesterase were performed under the same conditions; all sugars were liberated within an hour. The samples were dialyzed against 0.05 M NaCl solution overnight before isoelectric focusing.
RESULTS
Enzyme Purfication A two-step purification scheme which involved gel filtration on Sephadex G-50 fine and affinity chromatography was employed. In the gel filtration the venom acetylcholinesterase was eluted in the highmolecular-weight fraction ( M , > 30000) at the void volume of the gel. At this step 6-fold purification was obtained (Table 1); 20 g of the starting material, desiccated cobra venom, yielded 2.6 g lyophilized acetylcholinesterase preparation with specific activity about 79 IU/mg, which was stored at 4°C till use. The portions of 200mg of that preparation were subjected to affinity chromatography. Nearly 90 (’g of the whole acetylcholinesterase activity remained tightly bound into the column after washing with starting buffer. This activity could not be eluted with 1 M NaCl solution, and 0.05 M solution ofcompetitive acetylcholinesterase inhibitor phenyltrimethylammonium iodide was used for desorption. In some cases acetylcholinesterase was eluted by pH change. Lowering the pH to 5.0 (0.1 M NaCHKOO, 0.2 M NaCl)
154
Cobra Venom Acetykholinesterase
caused complete elution of the enzyme from the column in a sharp peak, but a lower yield and final purification rate were obtained. As a result of the purification, a preparation with a specific activity of 5000 IU/mg, as measured with acetylcholine on the pH-stat, was obtained. Concentrated acetylcholinesterase preparation (0.5-2.0 mg/ml) was stable for at least 2 months at 4 C in 0.05 M phosphate buffer, at pH 7.6, 0.2 M NaCI. For longer storage periods the solution was kept frozen at - 30 "C. A hsorpt ion Co&cient arid Sicllic Acid Content Determination
An approximate value of A;$ = 16.2 cm-' was obtained for pure acetylcholinesterase. Protein concentration was estimated according to Hartree [21]. Sialic acid determination with acetylcholinesterase gave the value 0.3 when N-acetylneuraminic acid was used as standard and the Lowry method was employed for protein concentration measurement. Assuming an acetylcholinesterase molecular weight of 67000, there is approximately 0.7 neuraminic acid residue per enzyme molecule.
:
Cobra Venom Acetylcholinesterase Purification and Molecular Properties Raivo RABA. Aavo AAVIKSAAR, Mari RABA, and Juri SIlGUR Biochemistry Department, Institute of Cybernetics of the Academy of Sciences of the Estonian SSR, Tallinn (Received August 14. 1978iJanuary 4, 1979)
Acetylcholinesterase from cobra (Nu@ naja oxiunu) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67000 i-2000. At high enzyme concentrations ( > 0.2 mg/ml, > 1 pM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1 -S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.
In recent years several acetylcholinesterases from animal tissues (electric organ of fishes, erythrocytes and brain of mammals etc.) have been purified up to a high degree of purity [I - 71. This has led to intensive work on the molecular structure of the enzyme. The investigations have elucidated a remarkable heterogeneity of acetylcholinesterases from various sources. In the case of electroplax there exist at least six forms of different size and shape [S]. Analogous multiple forms have been obtained for the erythrocyte enzyme [9,10]. In the detergent-free medium mammalian acetylcholinesterase also irreversibly forms aggregates of different molecular weights [ll]. In addition, as has been confirmed by Brodbeck et al. [12], any of these multiple forms of electric organ and erythrocyte acetylcholinesterase can be separated further into several fractions by isoelectric focusing. Although the catalytic specificity of Elapidae venom acetylcholinesterases has been shown to coincide with the specificity of the erythrocyte enzyme [13- 161, the venom enzymes seem to have a much less complicated structure than the acetylcholinesterase from membranes. Kumar and Elliott [5] who have investigated Bungurus,fizsciutus venom acetylG i : j . , i i e . s . Acetylcholinesterase o r acetylcholine acetyl-hydrolase (EC 3.1.1.7): ncuraminidase (EC 3.2.1.18). R’c~tc.This is the third paper in a series on C O ~ TVCPKJVI LI ACCIVIc./io/i~7r,.sir,.trsc.:the second paper appeared elsewhere [14].
cholinesterase, found only a single dimeric form of the enzyme with the molecular weight of 126000, consisting of identical catalytically active subunits. In a more recent work Lee, Latta and Elliott [17] reported that they have found from three elapid venoms several acetylcholinesterase isoenzymes differing in charge. Considering the similarities in catalytic properties of venom and tissue acetylcholinesterases we assume that the venom enzyme could serve as a convenient model for studying acetylcholinesterase mechanisms. In this work we describe the purification of cobra (Nuju nuju oxiunu) venom acetylcholinesterase. Molecular properties of the enzyme have been investigated and compared with other acetylcholinesterases.
MATERIALS AND METHODS Muteriuls
Desiccated Middle-Asian cobra (Nuju naju o.xiunu) venom was purchased from the Taschkent Integrated Zoo Plant (Uzbek SSR). Marker proteins used were : ovalbumin, catalase, chymotrypsinogen (all from Reanal, Hungary), cytochrome c (Serva, Heidelberg), cc-chymotrypsin (Worthington) and bovine serum albumin (Koch-Light laboratories, Colnbrook).
152
Cobra Venom Acetylcholinesterase
Neuraminidase (Vibrio cholerae, grade B, 500 U/ mg) was obtained from Calbiochem. Ampholine polyacrylamide gel plates pH 3.5- 10, were supplied by LKB Producter AB (Bromma). AH-Sepharose4B, Sephadex G-50 f, Sephadex G-100 sf and blue dextran 2000 were products of Pharmacia Fine Chemicals (Uppsala). Coomassie brilliant blue R250 ((2.1. 42660) was obtained from Ferak, fast blue RR salt from Reanal (Hungary) and basic fuchsin ((2.1. 42500) from Reachim (USSR). All other reagents used were of analytical grade.
Synthesis of Aflinity Adsorbent
N , N , N - Trimethyl - (m- aminophenyl) -ammonium chloride was synthesized according to Traylor and Singer [18]. This compound was quite a strong inhibitor for cobra venom acetylcholinesterase (Ki = 0.1 mM at 25 "C, pH 7.5, acetylcholine as substrate). Succinylated AH-Sepharose was prepared as described by Cuatrecasas and inhibitor was attached to the arm by carbodiimide coupling [19]. The affinity adsorbent had the following structure Sepharose 4B NH
-
(CH2)6- N H -CO
-
Assay of' Acetylcholinesterase Activity In the course of purification, acetylcholinesterase activity was determined spectrophotometrically with acetylthiocholine in the presence of a chromogenic agent 5,5'-dithio-bis(2-nitrobenzoic)acid as described by Ellmann et al. [20]. The reaction was carried out at room temperature on Specord UV-VIS spectrophotometer (Carl Zeiss, Jena). Acetylcholinesterase activity determination with acetylcholine was performed with the pH-stat method on a Radiometer TTT2/SBR3/ABU12 Titrigraph set. The reaction mixture contained 0.1 M NaCl, 0.01 M MgC12 and 1 mM acetylcholine. Acetylcholine hydrolysis was carried out at 25 "C and pH 7.5 under a stream of (202-free air. Activity of the enzyme was expressed in the international activity units (IU, pmol min- '). Activity localization in electrophoresis and isoelectric focusing experiments was performed by two different methods. The sensitive, although less specific, method of esterase activity staining with a-naphthyl acetate using the azo-coupling reagent fast blue RR salt was usually preferred. As established by Mounter [13], cobra venom does not contain any other enzyme
(CH2)2 - CO - NH -
Enzyme Purification All purification procedures were carried out at 4°C. Step I : Gel Filtration. 20 g of crude venom was dissolved in 0.2 M ammonium acetate pH 6.8, centrifuged to remove insoluble contaminants and passed through a column (110 x 11 cm) of Sephadex G-50 fine. Fractions containing acetylcholinesterase activity were pooled together and lyophilized. Lyophilized protein was stored at 4°C until use (up to 6 months). Step I I : Affinity Chromatography. 20 ml of affinity gel prepared as described was packed into a 14 x 1.4-cm column and equilibrated with 0.1 M sodium bicarbonate buffer pH 7.9, containing 0.2 M NaCl and 0.01 M MgC12. 100- 200 mg of acetylcholinesterase preparation from step I was dissolved in the elution buffer and dialyzed overnight. The sample was applied into the column and unadsorbed material was eluted with the buffer. For acetylcholinesterase elution, 0.05 M phenyltrimethylammonium iodide solution in the elution buffer was used. Eluted acetylcholinesterase was collected, concentrated by dialysis against 10 '%; poly(ethy1ene glycol) solution in 0.2 M NaCl, and finally dialysed against desired medium. Enzyme was usually stored in 0.1 M phosphate buffer pH 7.5, 0.2 M NaCI, at 4"C, or at -30°C for periods longer than one month. N o detectable loss of activity was observed under these conditions.
(0) Y N(CH3)3. +
capable of hydrolysing carboxylate esters. The gel was incubated after electrophoresis with 0.2 M phosphate buffer pH 7.0, for 5 - 10 min, then 4 ml of 0.5% a-naphthyl acetate and 2.5% fast blue RR salt solution in 50% acetone was added per 100 ml of the buffer solution, and mixed quickly. The reaction mixture was incubated for 5-30 min, depending on enzyme concentration. Alternatively, acetylcholinesterase was developed with acetylthiocholine according to Tripathi and O'Brien [7]. This method is specific and highly sensitive but the opaque enzyme bands obtained were less stable and produced poor photographic images. Protein concentration was determined by the modified Lowry method according to Hartree [21] using bovine serum albumin as standard.
Sedimentation Analysis The analysis was performed on Beckman Spinco E ultracentrifuge equipped with schlieren optics. The acetylcholinesterase sample was run at 60000 rev./min and 21 "C in 0.16 M NaCl at two different enzyme concentrations (2.8 and 1.4 mgiml). The values of were calculated in both cases. s20.
EIectroplzorc.sis Sodium dodecylsulfate/polyacrylamide gel electrophoresis was carried out in a discontinuous buffer
153
R. Raba, A. Aaviksaar, M. Raba, and J. Siigur Table 1 Purification of Naja naja oxiana venom acetylcholinrsteruse Step I1 was carried out on 200 mg of material from step I; the results obtained have been adjusted proportionately Total protein
Fraction
Total activity
Specific activity
Purification ~
~
~
~
.
overall
Crude venom I Gel filtration I1 Affinity chromatography
mg
IU
IU/mg
-fold
20000 2 635 34
263000 208 000 173900
13 1 78 8 5000
6 380
system according to Laemmli [22]. The gel tubes were stained immediately after electrophoresis with 0.1 Coomassie brilliant blue R250 solution in water/ ethano1:acetic acid mixture (7/7/1) for 2 h, destained with 7 (’( acetic acid in 25 ethanol and stored in 7 acetic acid. Disc electrophoresis was performed according to Ornstein and Davis in 7.5% gel at pH 8.3 [23]. The gel tubes were stained and destained as described by Chrambach et al. [24]. For glycoprotein staining, the periodic acid/Schiff reagent procedure described by Anselstetter and Horstmann was used 1251.
x
x
Gel Filtration For molecular weight determination, Sephadex G-100sf gel filtration media were used. The gel was packed into a column of 1.2 x 65 cm; 25 mM phosphate buffer solution pH 7.0 containing 0.1 M NaCl and 0.02”(; NaN3 was used for elution at a flow rate of 2.5 ml/h. Bovine serum albumin, ovalbumin and chymotrypsinogen were used as standard proteins. All the proteins were separately applied in 1-ml volume at 1 -2-mg/ml concentration into the column, and the column eluate was monitored by LKB Uvicord 111 analyzer at 281 nm to determine elution volume V,. Blue dextran 2000 was used as a void volume (VO) marker. A log M , versus Ve/Vo plot was used as a calibration curve in the molecular weight determination of acetylcholinesterase.
Isoelectric Focusing Analytical electrofocusing experiments were performed with LKB Multiphor apparatus using standard polyacrylamide gel plates, pH 3.5- 10. The plates were fixed and stained for protein as recommended by the manufacturer, LKB Producter AB (Bromma). Staining of the gel for enzyme activity was performed as described above. The pH on the plate surface was measured by means of a calibrated antimony/calomel electrode pair [26].
Recovery .~
.~ .~ .
..
. .
.
over preceding step
overall
over preceding step
6 63 5
79 66
19 84
Sialic Acid Determination and Neuraminidase Treatment Sialic acid content in the acetylcholinesterase molecule was determined according to Warren [27]. N-Acetylneuraminic acid was used as a standard. Neuraminidase treatment was carried out with Vibrio cholerae neuraminidase. To 0.4mlO. 1 M sodium acetate buffer (pH 5.5, 0.2 M NaCl, 0.02 M CaCb), 0.1 ml acetylcholinesterase solution (2 mg/ml, 0.2 M NaCl) and 0.1 ml neuraminidase solution (500 Ujml) were added. The mixture was incubated for 24 h or 96 h at 30 “C. Control experiments with human serum instead of acetylcholinesterase were performed under the same conditions; all sugars were liberated within an hour. The samples were dialyzed against 0.05 M NaCl solution overnight before isoelectric focusing.
RESULTS
Enzyme Purfication A two-step purification scheme which involved gel filtration on Sephadex G-50 fine and affinity chromatography was employed. In the gel filtration the venom acetylcholinesterase was eluted in the highmolecular-weight fraction ( M , > 30000) at the void volume of the gel. At this step 6-fold purification was obtained (Table 1); 20 g of the starting material, desiccated cobra venom, yielded 2.6 g lyophilized acetylcholinesterase preparation with specific activity about 79 IU/mg, which was stored at 4°C till use. The portions of 200mg of that preparation were subjected to affinity chromatography. Nearly 90 (’g of the whole acetylcholinesterase activity remained tightly bound into the column after washing with starting buffer. This activity could not be eluted with 1 M NaCl solution, and 0.05 M solution ofcompetitive acetylcholinesterase inhibitor phenyltrimethylammonium iodide was used for desorption. In some cases acetylcholinesterase was eluted by pH change. Lowering the pH to 5.0 (0.1 M NaCHKOO, 0.2 M NaCl)
154
Cobra Venom Acetykholinesterase
caused complete elution of the enzyme from the column in a sharp peak, but a lower yield and final purification rate were obtained. As a result of the purification, a preparation with a specific activity of 5000 IU/mg, as measured with acetylcholine on the pH-stat, was obtained. Concentrated acetylcholinesterase preparation (0.5-2.0 mg/ml) was stable for at least 2 months at 4 C in 0.05 M phosphate buffer, at pH 7.6, 0.2 M NaCI. For longer storage periods the solution was kept frozen at - 30 "C. A hsorpt ion Co&cient arid Sicllic Acid Content Determination
An approximate value of A;$ = 16.2 cm-' was obtained for pure acetylcholinesterase. Protein concentration was estimated according to Hartree [21]. Sialic acid determination with acetylcholinesterase gave the value 0.3 when N-acetylneuraminic acid was used as standard and the Lowry method was employed for protein concentration measurement. Assuming an acetylcholinesterase molecular weight of 67000, there is approximately 0.7 neuraminic acid residue per enzyme molecule.
:
