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Qualitative separation of choline esters by means of high voltage paper electrophoresis
SI-IORTCOMMUNICATIONS
257
1 c. MICWALEC,
Naturwissenschafteut, 42 (1955) 509. Biochim. Biophys. Acta, 19 (1956) 187. 3 c. MICHALEC, I+ater?z. Conf. on Biockem. Pvtiblems of Lipids, 4tJb Conf., Oxfovd, July 4 c, MICWALEC,V. JIRGL AND J. PODZIMEK, Experieuttia, 13 (1957) 342. 6J.A. LABARRBRE, J.R.CHIPAULT ANDW. 0. LUNDBERG, AnaLChem., 30 (1958) ?M.L. QUAIFPE, R.P. GEYER AND H.R.BOLLIGER, Anal. CJrem., 31 (1959) 950. 7 P. W. HANSEN AND H. DAM, AG~LZCl&em. Scan&,,11 (1957) 1658, * F. BoDE,A~Y&Z. Lab., 5 (1959) 221. 0 G. ZIMMERMANN AND G.BRKsE, Plzarmazie, II (1956) 715.. 10 I. M. PAGE AND N. RUDY, BiocJ&em.Z., 220 (1930) 304. 1pL. SWELL AND C. R.TREADWELL,J. BioZ,CAc?n.,212 (19553 141. c 2 c. MICHALBC.
Received
April
27th
x5-18,
1957.
1466.
1~~60 J. CJ&vomatog.,
Qualitative high
separation voltage
of choline paper
esters
by means
4 (1960)
254-257
of
electrophoresis
Choline esters occur in living material and these compounds as well as several synthetic choline esters are of considerable pharmacological interest. Separation and identification is often effected by means of paper chromatography. Since this procedure takes rather a long time, hydrolysis of the esters may easily occur. Therefore the authors have for some time used high voltage paper electrophoresis. The method has been described earlier by one ,of the authors in connection with studies on thiocholine estersl. About 20 choline esters and y-aminobutyric acid have now been investigated as regards their behaviour in various buffer solutions, various staining methods having been used. E,?cperimelztaZ High voltage paper electrophoresis. The esters were dissolved in distilled water and applied with a micropipette. Paper electrophoresis was carried out as described earlier?, usually at a voltage of 1,500 V, and 30 V/cm. Thirty to sixty minutes were sufficient for the electrophoresis.The temperature of the paper was 12-13~ during electrophoresis Btifler sol&ions. The following buffer systems were applied: Triethylamine-CO,, 0.05 M, pH 9.1. Acetate-acetic acid, 6.20 M, pH 5.0. Acetate-acetic acid, 0.20 M, pH 4.6. Boric acid-sodium hydroxide, 0.10 M, pH 10.0. Identification. After electrophoresis the papers were air-dried. For detection of the spots the following methods were used: Quaternary ammonium was identified with dipicrylamine2 or with phosphomolybdic acid3. In some cases iron-hydroxamate4 was used. y-Aminobutyric acid was detected with ninhydrin. Results Table I shows the mobilities relative to choline of a number of choline esters in various buffer systems. Separations of mixtures of acetylcholine, propionylch&ne and ‘,
I( .
_.. .* ”
J. Clrromatog.,
4 (ig6o)z57-259
SHORT COMMUNICATIONS
258
butyrylcholine and of y-aminobutyric acid, y-aminobutyrylcholine, acetylcholine and choline were carried out (see Fig. I). y-Aminobutyric acid showed tailing. The reason for this is probably dissociation of the acid. The relatively small difference between the mobility of y-aminobutyric acid at pH 4.6 and that at pH 10.0 may be explained by the values of the dissociation.constants of the acidfi; at IoOPI<~ (-COOH) was found to be 4.05 and pK, (-NH,+) was 11.02. The amount of choline esters most suitable for detection with dipicrylamine or phosphomolybdic acid is I-IO y per spot. Amounts down to 0.5 y could be detected in the case of a few substances, e.g. succinylcholine. TABLE MOBILITIES
OF
Migration
SOME
CHOLINE
rates are relative
ESTERS
IN
I POUR
DIl?FERENT
Buffer
Acetylcholinc
iodide
Propionylcholine’ Butyrylcholine’
iodide
SOLUTIONS
soldons
& 0.05
0.91
-&
0.04
0.89
f
0.01
o.gG & 0.03
0.75) =t 0.03
0.87
f
0.01
0.84
f
0.01
o.sg
0.70
& 0.02
0.50
Ifi
0.01
0.53
zt 0.01
0.82
0.70
f
0.05
0172 -J=0.06
o.so
& 0.01
0.74
& 0.06
0.79
f
0.53
*
0.02
0.90 -4:0.02
o.sg
&
0.01
-
0.89
iodide
BUFFER
to the rate of choline chloride or choline iodide.
It-Valerylcholine
iodicle
0.69
f
Isovalerylcholinc
iodide
0.69
& 0.03
O.SI
& 0.03
0.04
-
f
0.02
0.02
Acetyl-a-mctl~ylcholine iodide Acetyl-P-methylcholine
0.85
&
0.02
iodide o.g3
-J_-0.01
0.91
0.69
& 0.05
0.71
& o.oJ.
0.76
&
0.02
0.8G & 0.02
o.so
& 6.01
0.79
& 0.01
0.85
f
0.03
0.85
-& 0.03
0.64
& 0.01
o&4
& 0.01
o.Gg & 0.05
0.79
f
0.70
f. 0.02
-
o.Gg & 0.04
0.81
f
0.04
1.08 f
0.01
0.95 f
0.03
0.73
0.02
Acetyl-~,/&dimethylcholinc iodide Acrylylcholine
iodide
/3,/3-Dimethylacrylylcl~olinc iodide
0.01
a-Methylcrotonylcholine iodide Succinylcholine
chloride
Phenylsuccinylcholine Benzoylcholine
iodide
chloride
Nicotinylcholino Myristylcholine
iodide iodide
1.02 f
0.05
1.63 f
0.02
1.01 =t 0.03
0.91
f
0.04
0.84
0.03
0.85
0.68
f
0.02
o,G6 -1: 0.04
0.6G f
0.02
o.G2
starting point
f
&
starting
0.04 point
& 0.03
f
o.Gg f
0.03
0.70
0.05
‘0.74 & 0.02
point
starting point
f
starting
/?-Biomethyltrimethylammonium
bromide
P-Methylcholine
o.gG f
iodide
0.02
0.8s
0.91
f -
0.01
&
0.12
0.93
rf= 0.03 -
0.97
f
0.02
0.96
rfr 0.02
y-Aminobutyrylcboline iodide
1.20 &
‘$Ainiriobu,tyric ."./ ,'..
,‘.
,,
.I,
acid
,/.
0.30
0.04
=t 0.10
1~14
-
1.14
1.08 0.12
&
0.64
,‘.;. -.
.(,.
o.‘&G & 0.08
’ ‘.
.' ,
J. Clwomatog.,
4 (1960) 257-259
SI-IORT COM&IUNICATIONS
259
The electrophoretograms show that’no hydrolysis occurs during the time necessary for electrophoresis. Since there is no great difference in the mobility of the choline esters in .the different buffer systems used, the authors recommend buffers of p1-E 4-5 for electrophoresis. It has been, shown, for instance by LAFSSON~, that the hydrolysis of choline esters is slowest at $H around 4. It has furthermore been observed that spots are easier to detect with buffers of lower pH, especially if dipicrylamine is used for staining,.because the background, the sprayed paper, is less coloured than at higher pH.
Fig. I. High volta@ paper electrophorcsis of a mixture of sLcetylcholineiodide (4), propionylcholinc iodide (G), butyrylcholine iodide (7) and choline chloride at pl-I 4,,9. A, B and I3 Frc pure subst,znces, C and E misturcs. It was found that different esters show a somewhat different colour when sprayed with dipicrylamine. The spots of acetylcholine and choline were blue-red while the spots of the other esters were orange-red. It has also been observed that phosphomolybdic acid is a more reliable developer than clipicrylamine, but dipicrylamine is somewhat more sensitive. The iron-hydrosamatc developer sometimes failed to give a coloured spot. Research Imtitute of National Defence, Stwdbyberg (Swedevz)
EDITIS HEILBRONN BLENDA
CARLSSON
1 E. HEILBRONN,A&Z 2 G. 3 C. 4V.
G E. 0 L.
Clzenz.Scalzd., IZ (1958) 1492. MALYOTH AND H. W. STEIN, Bz'ocl&em.Z., 323 (1952) zG5. LEVINE AND E. CI-IARGAFF,J. Viol. Clrem., xgz (1951) 465. P. WHITTAKER, Biockem.J., 51 (1952) 345. J. ICING, J. Ant. Chcm. SOIL, 76 (1954) 1006. LARSSON, Ada Chsnt. Scam?., 8 (1954) 1017.
Received
May 3rd, rg6o J. Chrowzaiog.,
4 (1960) 257~259
257
1 c. MICWALEC,
Naturwissenschafteut, 42 (1955) 509. Biochim. Biophys. Acta, 19 (1956) 187. 3 c. MICHALEC, I+ater?z. Conf. on Biockem. Pvtiblems of Lipids, 4tJb Conf., Oxfovd, July 4 c, MICWALEC,V. JIRGL AND J. PODZIMEK, Experieuttia, 13 (1957) 342. 6J.A. LABARRBRE, J.R.CHIPAULT ANDW. 0. LUNDBERG, AnaLChem., 30 (1958) ?M.L. QUAIFPE, R.P. GEYER AND H.R.BOLLIGER, Anal. CJrem., 31 (1959) 950. 7 P. W. HANSEN AND H. DAM, AG~LZCl&em. Scan&,,11 (1957) 1658, * F. BoDE,A~Y&Z. Lab., 5 (1959) 221. 0 G. ZIMMERMANN AND G.BRKsE, Plzarmazie, II (1956) 715.. 10 I. M. PAGE AND N. RUDY, BiocJ&em.Z., 220 (1930) 304. 1pL. SWELL AND C. R.TREADWELL,J. BioZ,CAc?n.,212 (19553 141. c 2 c. MICHALBC.
Received
April
27th
x5-18,
1957.
1466.
1~~60 J. CJ&vomatog.,
Qualitative high
separation voltage
of choline paper
esters
by means
4 (1960)
254-257
of
electrophoresis
Choline esters occur in living material and these compounds as well as several synthetic choline esters are of considerable pharmacological interest. Separation and identification is often effected by means of paper chromatography. Since this procedure takes rather a long time, hydrolysis of the esters may easily occur. Therefore the authors have for some time used high voltage paper electrophoresis. The method has been described earlier by one ,of the authors in connection with studies on thiocholine estersl. About 20 choline esters and y-aminobutyric acid have now been investigated as regards their behaviour in various buffer solutions, various staining methods having been used. E,?cperimelztaZ High voltage paper electrophoresis. The esters were dissolved in distilled water and applied with a micropipette. Paper electrophoresis was carried out as described earlier?, usually at a voltage of 1,500 V, and 30 V/cm. Thirty to sixty minutes were sufficient for the electrophoresis.The temperature of the paper was 12-13~ during electrophoresis Btifler sol&ions. The following buffer systems were applied: Triethylamine-CO,, 0.05 M, pH 9.1. Acetate-acetic acid, 6.20 M, pH 5.0. Acetate-acetic acid, 0.20 M, pH 4.6. Boric acid-sodium hydroxide, 0.10 M, pH 10.0. Identification. After electrophoresis the papers were air-dried. For detection of the spots the following methods were used: Quaternary ammonium was identified with dipicrylamine2 or with phosphomolybdic acid3. In some cases iron-hydroxamate4 was used. y-Aminobutyric acid was detected with ninhydrin. Results Table I shows the mobilities relative to choline of a number of choline esters in various buffer systems. Separations of mixtures of acetylcholine, propionylch&ne and ‘,
I( .
_.. .* ”
J. Clrromatog.,
4 (ig6o)z57-259
SHORT COMMUNICATIONS
258
butyrylcholine and of y-aminobutyric acid, y-aminobutyrylcholine, acetylcholine and choline were carried out (see Fig. I). y-Aminobutyric acid showed tailing. The reason for this is probably dissociation of the acid. The relatively small difference between the mobility of y-aminobutyric acid at pH 4.6 and that at pH 10.0 may be explained by the values of the dissociation.constants of the acidfi; at IoOPI<~ (-COOH) was found to be 4.05 and pK, (-NH,+) was 11.02. The amount of choline esters most suitable for detection with dipicrylamine or phosphomolybdic acid is I-IO y per spot. Amounts down to 0.5 y could be detected in the case of a few substances, e.g. succinylcholine. TABLE MOBILITIES
OF
Migration
SOME
CHOLINE
rates are relative
ESTERS
IN
I POUR
DIl?FERENT
Buffer
Acetylcholinc
iodide
Propionylcholine’ Butyrylcholine’
iodide
SOLUTIONS
soldons
& 0.05
0.91
-&
0.04
0.89
f
0.01
o.gG & 0.03
0.75) =t 0.03
0.87
f
0.01
0.84
f
0.01
o.sg
0.70
& 0.02
0.50
Ifi
0.01
0.53
zt 0.01
0.82
0.70
f
0.05
0172 -J=0.06
o.so
& 0.01
0.74
& 0.06
0.79
f
0.53
*
0.02
0.90 -4:0.02
o.sg
&
0.01
-
0.89
iodide
BUFFER
to the rate of choline chloride or choline iodide.
It-Valerylcholine
iodicle
0.69
f
Isovalerylcholinc
iodide
0.69
& 0.03
O.SI
& 0.03
0.04
-
f
0.02
0.02
Acetyl-a-mctl~ylcholine iodide Acetyl-P-methylcholine
0.85
&
0.02
iodide o.g3
-J_-0.01
0.91
0.69
& 0.05
0.71
& o.oJ.
0.76
&
0.02
0.8G & 0.02
o.so
& 6.01
0.79
& 0.01
0.85
f
0.03
0.85
-& 0.03
0.64
& 0.01
o&4
& 0.01
o.Gg & 0.05
0.79
f
0.70
f. 0.02
-
o.Gg & 0.04
0.81
f
0.04
1.08 f
0.01
0.95 f
0.03
0.73
0.02
Acetyl-~,/&dimethylcholinc iodide Acrylylcholine
iodide
/3,/3-Dimethylacrylylcl~olinc iodide
0.01
a-Methylcrotonylcholine iodide Succinylcholine
chloride
Phenylsuccinylcholine Benzoylcholine
iodide
chloride
Nicotinylcholino Myristylcholine
iodide iodide
1.02 f
0.05
1.63 f
0.02
1.01 =t 0.03
0.91
f
0.04
0.84
0.03
0.85
0.68
f
0.02
o,G6 -1: 0.04
0.6G f
0.02
o.G2
starting point
f
&
starting
0.04 point
& 0.03
f
o.Gg f
0.03
0.70
0.05
‘0.74 & 0.02
point
starting point
f
starting
/?-Biomethyltrimethylammonium
bromide
P-Methylcholine
o.gG f
iodide
0.02
0.8s
0.91
f -
0.01
&
0.12
0.93
rf= 0.03 -
0.97
f
0.02
0.96
rfr 0.02
y-Aminobutyrylcboline iodide
1.20 &
‘$Ainiriobu,tyric ."./ ,'..
,‘.
,,
.I,
acid
,/.
0.30
0.04
=t 0.10
1~14
-
1.14
1.08 0.12
&
0.64
,‘.;. -.
.(,.
o.‘&G & 0.08
’ ‘.
.' ,
J. Clwomatog.,
4 (1960) 257-259
SI-IORT COM&IUNICATIONS
259
The electrophoretograms show that’no hydrolysis occurs during the time necessary for electrophoresis. Since there is no great difference in the mobility of the choline esters in .the different buffer systems used, the authors recommend buffers of p1-E 4-5 for electrophoresis. It has been, shown, for instance by LAFSSON~, that the hydrolysis of choline esters is slowest at $H around 4. It has furthermore been observed that spots are easier to detect with buffers of lower pH, especially if dipicrylamine is used for staining,.because the background, the sprayed paper, is less coloured than at higher pH.
Fig. I. High volta@ paper electrophorcsis of a mixture of sLcetylcholineiodide (4), propionylcholinc iodide (G), butyrylcholine iodide (7) and choline chloride at pl-I 4,,9. A, B and I3 Frc pure subst,znces, C and E misturcs. It was found that different esters show a somewhat different colour when sprayed with dipicrylamine. The spots of acetylcholine and choline were blue-red while the spots of the other esters were orange-red. It has also been observed that phosphomolybdic acid is a more reliable developer than clipicrylamine, but dipicrylamine is somewhat more sensitive. The iron-hydrosamatc developer sometimes failed to give a coloured spot. Research Imtitute of National Defence, Stwdbyberg (Swedevz)
EDITIS HEILBRONN BLENDA
CARLSSON
1 E. HEILBRONN,A&Z 2 G. 3 C. 4V.
G E. 0 L.
Clzenz.Scalzd., IZ (1958) 1492. MALYOTH AND H. W. STEIN, Bz'ocl&em.Z., 323 (1952) zG5. LEVINE AND E. CI-IARGAFF,J. Viol. Clrem., xgz (1951) 465. P. WHITTAKER, Biockem.J., 51 (1952) 345. J. ICING, J. Ant. Chcm. SOIL, 76 (1954) 1006. LARSSON, Ada Chsnt. Scam?., 8 (1954) 1017.
Received
May 3rd, rg6o J. Chrowzaiog.,
4 (1960) 257~259