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Separation of adenosine 3′,5′-phosphata derivatives on thin layers of ECTEOLA cellulose
ANALYTICAL
20,
BIOCHEMISTRY
Separation
192-194
of Adenosine on Thin
Layers MARTIN
The
Rockefeller
University,
(1967)
3’,5’-Phosphate of ECTEOLA
Cellulose
A. RIZACK New
Received February
This phate activity layers similar
Derivatives
method was developed for the (cyclic-AMP) and its derivatives by scintillation counting. The of ECTEOLA cellulose spread to silica gel (1). EXPERIMENTAL
York,
New
York
10091
20, 1967
isolation of adenosine 3’,5’-phosand determination of their radioseparation is performed on thin on vinyl sheeting in a manner PROCEDURES
Materials. ECTEOLA cellulose for thin-layer chromatography was obtained from Schleicher and Schuell (Selectacel ,#67, capacity 0.24 meq/gm). Tritiated cyclic-AMP was obtained from Schwarz BioResearch and other compounds from Nutritional Biochemical. Bakelite (Union Carbide) rigid vinyl calendered sheeting was obtained in 20 X 50 in. sheets, which were cut to 8 X 50 in. on a band saw. Spreading. A slurry of ECTEOLA was prepared by dispersing 13 gm in 65 ml of distilled water with a semimicro container of a Waring Blendor. The vinyl sheeting was thoroughly washed in detergent and rinsed first in distilled water and then in ethyl alcohol. The ECTEOLA slurry was spread on the dried vinyl sheeting with a variable aperture spreader set at 0.25 mm. The air-dried thin layers were cut into 8 X 8 in. sections with a paper cutter and handled like glass plates. Chromatography. The chromatograms were run in two directions. First distilled water was allowed to ascend to the edge of the plate separating nucleotides from nucleosides and purine derivatives (2). The chromatogram was then developed in the second direction with citrate buffer, 0.035 M at pH 5’.5, after the impurities which accumulated at the front had been scraped off. Counting. Nonradioactive compounds were added to all samples at the origin of the chromatogram to function as both carrier and chromatographic standard. Compounds to be assayed for radioactivity were identified by locating the appropriate absorbing spot under short-wave ultraviolet light. The spots were then cut out with a scissors and placed 192
SEPARATIOPi
OF
CYCLIC-AMP
193
DERIV.l’I‘IVE~
in a scintillation vial. Sodium hydroxide (0.8 ml of 0.1 N) was added to elute the compounds from the ECTEOLA and 18 ml of scintillation fluid was added directly to this. The scintillation fluid contained 4 gm PPO/liter and 50 mg POPOP/liter in 50% toluene and 50% ethoxyethanol (v/v). It was not found necessary to remove the thin layer antI its supporting vinyl sheeting before adding the fluid. RESULTS
The separation obtained is shown in Figure 1. ATP and ilDP arc> separable from cyclic-AMP in this system since they run ahead of ,4MP in citrate buffer. ATP and ADP run together with an Rf of 0.61. while the Rf for AMP is 0.56. The capacity of one of these layers was approximately 6 pg applied to the origin. The application of 2 pg was required to visualize AMP under ultraviolet light, while only 1 pg of the other compounds was SOLVENT
FRONT -11
00
0 Adenosine
AMP
lnosine
Orrgin
Distilled
Water
-
FIG. 1. Tracing of chromatographic separation of cyclic-AMP ECTEOLA cellulose thin layer. Solvent front I (distilled water) bhe origin and solvent front II (0.035M ritrate buffer nt pH from the origin.
derivatives was 17.9 5.5) was
on an
cm from 16.2
rm
194
MARTIN
A.
RIZACK
necessary. Overloading caused tailing and incomplete separation of the compounds. Immersion of chromatograms contaminated the developing solution. Fresh solutions were therefore prepared for each run to prevent impairment of the degree of separation. The radioactivity recovered was 8%85% of that added to the chromatogram. The ECTEOLA did not cause quenching or self-absorption. The presence of the vinyl sheeting in the scintillation vials reduced the total counts by 2-3s. DISCUSSION
The method is being used for two purposes: a study of the metabolic products of cyclic-AMP in adipose tissue extracts and an assay for cyclic 3’,5’-nucleotide phosphodiesterase in adrenal, liver, and adipose tissue extracts. The complete separations obtained make it possible to recover any given compound for radioactive assay without contamination by other intermediates. The use of a buffer as a developing solution rather than the acid solutions used previously made it possible to control pH more precisely and thus enhance the degree of separation obtainable. An aqueous system is essential because vinyl sheeting has limited resistance to organic solvents. SUMMARY
Thin layers of ECTEOLA on vinyl sheeting were used to separate derivatives of cyclic-AMP. The complete separation of the compounds studied (cyclic-AMP, AMP, adenosine, inosine, and hypoxanthine) made it possible simply to cut the area containing any given compound from the chromatogram. With the use of labeled precursors the incorporation of radioactivity into the various compounds could be determined by eluting with alkali in a scintillation vial and adding scintillation fluid without removing the chromatographic medium. ACKNOWLEDGMENT The excellent knowledged. This research 1835.
technical was
assistance
supported
hy
of the
Mrs. National
REFERENCES 1. ~-SQUIBB, R. 2. RANDEFLATH, 3. RANDERATH,
L., K., K.,
Nature Angew.
Angew.
198, 317 (1963). Chem. 73, 674 (1961). Chem. 74, 484 (1962).
Virginia Science
Martin
is
Foundation
gratefully Grant
acGB
20,
BIOCHEMISTRY
Separation
192-194
of Adenosine on Thin
Layers MARTIN
The
Rockefeller
University,
(1967)
3’,5’-Phosphate of ECTEOLA
Cellulose
A. RIZACK New
Received February
This phate activity layers similar
Derivatives
method was developed for the (cyclic-AMP) and its derivatives by scintillation counting. The of ECTEOLA cellulose spread to silica gel (1). EXPERIMENTAL
York,
New
York
10091
20, 1967
isolation of adenosine 3’,5’-phosand determination of their radioseparation is performed on thin on vinyl sheeting in a manner PROCEDURES
Materials. ECTEOLA cellulose for thin-layer chromatography was obtained from Schleicher and Schuell (Selectacel ,#67, capacity 0.24 meq/gm). Tritiated cyclic-AMP was obtained from Schwarz BioResearch and other compounds from Nutritional Biochemical. Bakelite (Union Carbide) rigid vinyl calendered sheeting was obtained in 20 X 50 in. sheets, which were cut to 8 X 50 in. on a band saw. Spreading. A slurry of ECTEOLA was prepared by dispersing 13 gm in 65 ml of distilled water with a semimicro container of a Waring Blendor. The vinyl sheeting was thoroughly washed in detergent and rinsed first in distilled water and then in ethyl alcohol. The ECTEOLA slurry was spread on the dried vinyl sheeting with a variable aperture spreader set at 0.25 mm. The air-dried thin layers were cut into 8 X 8 in. sections with a paper cutter and handled like glass plates. Chromatography. The chromatograms were run in two directions. First distilled water was allowed to ascend to the edge of the plate separating nucleotides from nucleosides and purine derivatives (2). The chromatogram was then developed in the second direction with citrate buffer, 0.035 M at pH 5’.5, after the impurities which accumulated at the front had been scraped off. Counting. Nonradioactive compounds were added to all samples at the origin of the chromatogram to function as both carrier and chromatographic standard. Compounds to be assayed for radioactivity were identified by locating the appropriate absorbing spot under short-wave ultraviolet light. The spots were then cut out with a scissors and placed 192
SEPARATIOPi
OF
CYCLIC-AMP
193
DERIV.l’I‘IVE~
in a scintillation vial. Sodium hydroxide (0.8 ml of 0.1 N) was added to elute the compounds from the ECTEOLA and 18 ml of scintillation fluid was added directly to this. The scintillation fluid contained 4 gm PPO/liter and 50 mg POPOP/liter in 50% toluene and 50% ethoxyethanol (v/v). It was not found necessary to remove the thin layer antI its supporting vinyl sheeting before adding the fluid. RESULTS
The separation obtained is shown in Figure 1. ATP and ilDP arc> separable from cyclic-AMP in this system since they run ahead of ,4MP in citrate buffer. ATP and ADP run together with an Rf of 0.61. while the Rf for AMP is 0.56. The capacity of one of these layers was approximately 6 pg applied to the origin. The application of 2 pg was required to visualize AMP under ultraviolet light, while only 1 pg of the other compounds was SOLVENT
FRONT -11
00
0 Adenosine
AMP
lnosine
Orrgin
Distilled
Water
-
FIG. 1. Tracing of chromatographic separation of cyclic-AMP ECTEOLA cellulose thin layer. Solvent front I (distilled water) bhe origin and solvent front II (0.035M ritrate buffer nt pH from the origin.
derivatives was 17.9 5.5) was
on an
cm from 16.2
rm
194
MARTIN
A.
RIZACK
necessary. Overloading caused tailing and incomplete separation of the compounds. Immersion of chromatograms contaminated the developing solution. Fresh solutions were therefore prepared for each run to prevent impairment of the degree of separation. The radioactivity recovered was 8%85% of that added to the chromatogram. The ECTEOLA did not cause quenching or self-absorption. The presence of the vinyl sheeting in the scintillation vials reduced the total counts by 2-3s. DISCUSSION
The method is being used for two purposes: a study of the metabolic products of cyclic-AMP in adipose tissue extracts and an assay for cyclic 3’,5’-nucleotide phosphodiesterase in adrenal, liver, and adipose tissue extracts. The complete separations obtained make it possible to recover any given compound for radioactive assay without contamination by other intermediates. The use of a buffer as a developing solution rather than the acid solutions used previously made it possible to control pH more precisely and thus enhance the degree of separation obtainable. An aqueous system is essential because vinyl sheeting has limited resistance to organic solvents. SUMMARY
Thin layers of ECTEOLA on vinyl sheeting were used to separate derivatives of cyclic-AMP. The complete separation of the compounds studied (cyclic-AMP, AMP, adenosine, inosine, and hypoxanthine) made it possible simply to cut the area containing any given compound from the chromatogram. With the use of labeled precursors the incorporation of radioactivity into the various compounds could be determined by eluting with alkali in a scintillation vial and adding scintillation fluid without removing the chromatographic medium. ACKNOWLEDGMENT The excellent knowledged. This research 1835.
technical was
assistance
supported
hy
of the
Mrs. National
REFERENCES 1. ~-SQUIBB, R. 2. RANDEFLATH, 3. RANDERATH,
L., K., K.,
Nature Angew.
Angew.
198, 317 (1963). Chem. 73, 674 (1961). Chem. 74, 484 (1962).
Virginia Science
Martin
is
Foundation
gratefully Grant
acGB