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Separation of ribonucleosides from deoxyribo-nucleosides and arabinonucleosides by thin-layer chromatography
474
SHORT COMMUNICATIONS ACKXOWLEDGMENTS
The authors Horikama and
wish to acknowledge Miss Nobuko Isobe.
the
able
technical
assistance
of Miss
Michiko
REFERENCES 1. OVODOV,
Y.
SOLOV'EVA,
2. PRIDHAM,
S., EVTUSHENKO, E. V., VASKOVSKY, T. F., J. Chromatog. 26, 111 (1967). J. B., An.aZ. Chem. 28, 1967 (1956).
V.
E.,
OVODOVA,
R.
G.,
AND
KYOKO HOTTA MASAHARU KUROKAWA The Kitasato Institute The Kitasato I’?~iversity Tokyo, Japan Received June 3,196s
Separation
of
Ribonucleosides
nucleosides and Thin-layer
from
Deoxyribo-
Arabinonucleosides Chromatography
by
In studies dealing with ribonucleotide reductase and with the metabolism of arabinosylcytosine, it. became desirable to develop a rapid method for the separation of ribonucleosides from their deoxyribosyl and arabinosyl analogs. Randerath’s method for the t,hin-layer chromatography of the phosphorylated derivatives on PEI-cellulose’ with solutions of LiCl and boric acid (1, 2) does not give good resolution of the free nucleosides. However, Khym and Cohn’s studies on the anion-exchange chromatography of sugars (3, 4) and the more recent finding (5) that arabinosyleytosine could be separated from cytidine on Dowex 1 borate but not on Dowex 1 bicarbonate suggested that, in the case of the nucleosides, it would be advantageous to convert PEI-cellulose to the borate form so that complexes with cis-glycols would be formed on the stationary phase. The present communication describes this procedure. Plastic sheets, 20 x 20 cm, coated with approximately 0.1 mm of PEIcellulose (MN-Polygram CEL-3OO-PEI, Macherey, Nagel & Co., Diiren, Germany) were obtained from Brinkmann Instruments, Inc. They were ’ Obtained ethyleneimine
by treating cellulose (PEI) hydrochloride.
(for
thin-layer
chromatography)
with
poly-
SHORT
475
COMMUKICATIOSS
washed with 10% NaCl and water (6). The chloride form of the anion exchanger was then converted to the borate form by soaking the dried sheets for 5 min in 750 ml of 0.4 M triethylammonium tetraborate (99 gm of boric acid and 112 ml of triethylamine in a total volume of 1 liter). Excess solution was allowed to drain, and the sheets were immersed without drying in about 2 liters of distilled water for 1 min, t,hen in 500 ml of methanol for 1 min, dried overnight, and stored below 0”. The nucleosides (10 nmoles in 0.5-l ~11 were applied 2 cm from the Iower edge of the sheet. Ascending development was carried out in closed tanks with either of the following mixtures: (1) 0.1 YM boric acid; (2) 0.02 M ammonium formate, pH 4.7/ethanol (l/l). After drying, the compounds were visualized as quenching spots under ultraviolet light. As shown in Table 1, the procedure gave an excellent separation of rihoR, Values
of Nucleosides
TABLE 1 on PEI-Borate-Cellulose R/ X 100 for nucleoside
Nucleoside
Deoxyadenosine Adenosine Deoxycytidine Cytidine Arabinosylcytosine Deoxyguanosine Guanosine Deoxyinosine Inosine Deoxyuridine Uridine Deoxythymidine Arahinosvluracil a (1) 0.1 M boric
acid;
Thin in solventa
Layers No.
(1)
(2)
56 4 79 8 74 45 1 60 2 SO 6 85 75
71 16 72 13 64 51 3 49 3 70 10 7s 64
(2) 0.02 M ammonium
formate,
pH 4.7/ethanol
(l/l).
from deoxyribo- and arabinonucleosides. With the original chloride form of PEI-cellulose, some lesser separation could be achieved in either 0.2 M boric acid/ethanol (l/l) or 0.04 M triethylammonium tetraborate/ethano1 (l/l), but the ribonucleoside spots were much less compact. The borate form of PEI-cellulose can also be used with nucleoside 5’-monophosphates, although in this case both ionic forms of the ion exchanger are about equivalent (Table 2). This result is in agreement with findings of Khym et al. (3, 4) in column chromatography on Dowex 1. In the assay of radioactive nucleosides, rectangles surrounding the quenching spots were cut out (6)) placed at the bottom of standard 20 ml
476
SHORT
R, Values
of Nucleoside
COMMUNICATIONS
TABLE 5’-Monophosphates
2 on PEI-Cellulose
Thin
Layers”
R, X100 Compound
PEI-borate
PEI-chloride
$2
26 5 20 3
dAMP AMP dCMP CMP dGMP GMP IMP dUMP UMP dTMP
23 48 216 30 5 14 50 21 55
a Development: 1.0 M LiCl saturated with (l/l). Aqueous mixtures produced considerable b Elongated spot.
IO
3 3 27 4 32
boric acid streaking
(adjust,ed to pH on PET-borate.
4.5)/ethanol
glass counting vials, and covered with 5 ml of a solution containing 5 gm of 2,5-diphenyloxazole and 0.3 gm of 1,4-bis-2- (4-methyl-5-oxazolyl) benzene per liter of toluene. Counting efficiency in a Beckman scintillation counter was about 85% in the case of ‘% and 15% in the case of 3H. The present technique can be applied advantageously to the measurement of ribonucleotide reduct.ase activity (D. W. Jacobsen and F. M. Huennekena, unpublished results). The ribo- and deoxyribonucleoside triphosphates (or diphosphates), which are present in the reaction mixture, are hydrolyzed with alkaline phosphatase (Escherichia cd) to the nucleosides (7), and the latter are then separated on PEI-borate-cellulose thin-layer sheets. This procedure is indicated especially in the case of adenine and guanine derivatives because acid hydrolysis to the monophosphates is inapplicable to purine nucleotides. The alternat.ive twodimensional thin-layer chromatography of the triphosphates (8, 9) produces less complete separation of the deoxyribo from the ribo compounds and requires one thin-layer sheet for each sample. ACKNOWLEDGMENT This study was supported in part by U. S. Public Health Research Grant CA-6522 from the National Cancer Institute and was carried out during a tenure of one of us (A.W.S.) as a Visiting Investigator at the Scripps Clinic and Research Foundation. REFERENCES 1. RANDERATH,
h-., Biochim.
2.
R.~NDERATH,
K.,
3.
KHYM,
J. X.,
AND AND
Biophys. Acta 76, 622 (1963). E.,J. Chromatog. L. P.. J. Am. Chew Sot.
RAXDERATH,
ZILL.
16, 111 (1964). 74, 2090 (1952).
SHORT
477
COMMUNICATIONS
4. KHYM,
J. X., AND COHN, W. E., J. Am. Chem. Sot. 75, 1153 (1953). A. IV., MEAD, J. A. R., AND URSHEL. M. J., Biochem. Plmmncol. 15, 1443 (1966). RANDERATH, K., AND R.~SDER.~TH, E.. J. Chu)nmfrjg. 22, 110 (1966). HEPPEL, L. A., HARKNESS, D. R., .4ND HILMOE, R. J.. J. Viol. Chem. 237, 541 (1962). GOULIAN, M., .~ND BECK, IV. S., J. Biol. Chem. 241, 4233 (1966). ~TEUHARD, D. J., RASDERATH, E., AND RANDEHATH, Ii., Ann2. Biochem. 13, 211 (1965).
5. SCHRECKER,
6. 7. 8.
9.
-ANTHONY National Natio,nal Bethesda,
W.
~CHRECKER
Cancer Iwtitute Institutes of Health Maryland 20014 DONALD *JULIUS
Biochemistry Department Scripps Clinic and Research La Jolla, California 92037 Received June +?S, 1968
A Low-Cost
W.
,JACOBSES KIRCHNER
Foundation
Variable-Proportion
Gas-Metering
System
Many laboratories employ gas mixtures in a variety of applications. Uses include gas chromatography, nuclear counter tubes, semiconductor doping, crystal growt,h, and a great variety of biological applications. When a single gas mixture is desired, most users prefer to purchase premixed gas. There are, however, two major disadvantages in purchasing premixed gas: the gas often must be special-ordered with a subsequent long delivery time, and the mixtures may vary slightly in composition from one tank to another or from the beginning to the end of the same tank. When the precise composition of the gas mixture must be varied, a gasmetering pump and cylinders of pure gasses are generally used. Such pumps are quite expensive-this is frequently sufficient to discourage some lines of investigation. Many pumps do not have adequate delivery precision at the lower flow rates required in many applications. Theory. Under conditions of laminar (nonturbulent) flow, the flow rate of a fluid through a tube may be predicted by the Poiseuille equation:
SHORT COMMUNICATIONS ACKXOWLEDGMENTS
The authors Horikama and
wish to acknowledge Miss Nobuko Isobe.
the
able
technical
assistance
of Miss
Michiko
REFERENCES 1. OVODOV,
Y.
SOLOV'EVA,
2. PRIDHAM,
S., EVTUSHENKO, E. V., VASKOVSKY, T. F., J. Chromatog. 26, 111 (1967). J. B., An.aZ. Chem. 28, 1967 (1956).
V.
E.,
OVODOVA,
R.
G.,
AND
KYOKO HOTTA MASAHARU KUROKAWA The Kitasato Institute The Kitasato I’?~iversity Tokyo, Japan Received June 3,196s
Separation
of
Ribonucleosides
nucleosides and Thin-layer
from
Deoxyribo-
Arabinonucleosides Chromatography
by
In studies dealing with ribonucleotide reductase and with the metabolism of arabinosylcytosine, it. became desirable to develop a rapid method for the separation of ribonucleosides from their deoxyribosyl and arabinosyl analogs. Randerath’s method for the t,hin-layer chromatography of the phosphorylated derivatives on PEI-cellulose’ with solutions of LiCl and boric acid (1, 2) does not give good resolution of the free nucleosides. However, Khym and Cohn’s studies on the anion-exchange chromatography of sugars (3, 4) and the more recent finding (5) that arabinosyleytosine could be separated from cytidine on Dowex 1 borate but not on Dowex 1 bicarbonate suggested that, in the case of the nucleosides, it would be advantageous to convert PEI-cellulose to the borate form so that complexes with cis-glycols would be formed on the stationary phase. The present communication describes this procedure. Plastic sheets, 20 x 20 cm, coated with approximately 0.1 mm of PEIcellulose (MN-Polygram CEL-3OO-PEI, Macherey, Nagel & Co., Diiren, Germany) were obtained from Brinkmann Instruments, Inc. They were ’ Obtained ethyleneimine
by treating cellulose (PEI) hydrochloride.
(for
thin-layer
chromatography)
with
poly-
SHORT
475
COMMUKICATIOSS
washed with 10% NaCl and water (6). The chloride form of the anion exchanger was then converted to the borate form by soaking the dried sheets for 5 min in 750 ml of 0.4 M triethylammonium tetraborate (99 gm of boric acid and 112 ml of triethylamine in a total volume of 1 liter). Excess solution was allowed to drain, and the sheets were immersed without drying in about 2 liters of distilled water for 1 min, t,hen in 500 ml of methanol for 1 min, dried overnight, and stored below 0”. The nucleosides (10 nmoles in 0.5-l ~11 were applied 2 cm from the Iower edge of the sheet. Ascending development was carried out in closed tanks with either of the following mixtures: (1) 0.1 YM boric acid; (2) 0.02 M ammonium formate, pH 4.7/ethanol (l/l). After drying, the compounds were visualized as quenching spots under ultraviolet light. As shown in Table 1, the procedure gave an excellent separation of rihoR, Values
of Nucleosides
TABLE 1 on PEI-Borate-Cellulose R/ X 100 for nucleoside
Nucleoside
Deoxyadenosine Adenosine Deoxycytidine Cytidine Arabinosylcytosine Deoxyguanosine Guanosine Deoxyinosine Inosine Deoxyuridine Uridine Deoxythymidine Arahinosvluracil a (1) 0.1 M boric
acid;
Thin in solventa
Layers No.
(1)
(2)
56 4 79 8 74 45 1 60 2 SO 6 85 75
71 16 72 13 64 51 3 49 3 70 10 7s 64
(2) 0.02 M ammonium
formate,
pH 4.7/ethanol
(l/l).
from deoxyribo- and arabinonucleosides. With the original chloride form of PEI-cellulose, some lesser separation could be achieved in either 0.2 M boric acid/ethanol (l/l) or 0.04 M triethylammonium tetraborate/ethano1 (l/l), but the ribonucleoside spots were much less compact. The borate form of PEI-cellulose can also be used with nucleoside 5’-monophosphates, although in this case both ionic forms of the ion exchanger are about equivalent (Table 2). This result is in agreement with findings of Khym et al. (3, 4) in column chromatography on Dowex 1. In the assay of radioactive nucleosides, rectangles surrounding the quenching spots were cut out (6)) placed at the bottom of standard 20 ml
476
SHORT
R, Values
of Nucleoside
COMMUNICATIONS
TABLE 5’-Monophosphates
2 on PEI-Cellulose
Thin
Layers”
R, X100 Compound
PEI-borate
PEI-chloride
$2
26 5 20 3
dAMP AMP dCMP CMP dGMP GMP IMP dUMP UMP dTMP
23 48 216 30 5 14 50 21 55
a Development: 1.0 M LiCl saturated with (l/l). Aqueous mixtures produced considerable b Elongated spot.
IO
3 3 27 4 32
boric acid streaking
(adjust,ed to pH on PET-borate.
4.5)/ethanol
glass counting vials, and covered with 5 ml of a solution containing 5 gm of 2,5-diphenyloxazole and 0.3 gm of 1,4-bis-2- (4-methyl-5-oxazolyl) benzene per liter of toluene. Counting efficiency in a Beckman scintillation counter was about 85% in the case of ‘% and 15% in the case of 3H. The present technique can be applied advantageously to the measurement of ribonucleotide reduct.ase activity (D. W. Jacobsen and F. M. Huennekena, unpublished results). The ribo- and deoxyribonucleoside triphosphates (or diphosphates), which are present in the reaction mixture, are hydrolyzed with alkaline phosphatase (Escherichia cd) to the nucleosides (7), and the latter are then separated on PEI-borate-cellulose thin-layer sheets. This procedure is indicated especially in the case of adenine and guanine derivatives because acid hydrolysis to the monophosphates is inapplicable to purine nucleotides. The alternat.ive twodimensional thin-layer chromatography of the triphosphates (8, 9) produces less complete separation of the deoxyribo from the ribo compounds and requires one thin-layer sheet for each sample. ACKNOWLEDGMENT This study was supported in part by U. S. Public Health Research Grant CA-6522 from the National Cancer Institute and was carried out during a tenure of one of us (A.W.S.) as a Visiting Investigator at the Scripps Clinic and Research Foundation. REFERENCES 1. RANDERATH,
h-., Biochim.
2.
R.~NDERATH,
K.,
3.
KHYM,
J. X.,
AND AND
Biophys. Acta 76, 622 (1963). E.,J. Chromatog. L. P.. J. Am. Chew Sot.
RAXDERATH,
ZILL.
16, 111 (1964). 74, 2090 (1952).
SHORT
477
COMMUNICATIONS
4. KHYM,
J. X., AND COHN, W. E., J. Am. Chem. Sot. 75, 1153 (1953). A. IV., MEAD, J. A. R., AND URSHEL. M. J., Biochem. Plmmncol. 15, 1443 (1966). RANDERATH, K., AND R.~SDER.~TH, E.. J. Chu)nmfrjg. 22, 110 (1966). HEPPEL, L. A., HARKNESS, D. R., .4ND HILMOE, R. J.. J. Viol. Chem. 237, 541 (1962). GOULIAN, M., .~ND BECK, IV. S., J. Biol. Chem. 241, 4233 (1966). ~TEUHARD, D. J., RASDERATH, E., AND RANDEHATH, Ii., Ann2. Biochem. 13, 211 (1965).
5. SCHRECKER,
6. 7. 8.
9.
-ANTHONY National Natio,nal Bethesda,
W.
~CHRECKER
Cancer Iwtitute Institutes of Health Maryland 20014 DONALD *JULIUS
Biochemistry Department Scripps Clinic and Research La Jolla, California 92037 Received June +?S, 1968
A Low-Cost
W.
,JACOBSES KIRCHNER
Foundation
Variable-Proportion
Gas-Metering
System
Many laboratories employ gas mixtures in a variety of applications. Uses include gas chromatography, nuclear counter tubes, semiconductor doping, crystal growt,h, and a great variety of biological applications. When a single gas mixture is desired, most users prefer to purchase premixed gas. There are, however, two major disadvantages in purchasing premixed gas: the gas often must be special-ordered with a subsequent long delivery time, and the mixtures may vary slightly in composition from one tank to another or from the beginning to the end of the same tank. When the precise composition of the gas mixture must be varied, a gasmetering pump and cylinders of pure gasses are generally used. Such pumps are quite expensive-this is frequently sufficient to discourage some lines of investigation. Many pumps do not have adequate delivery precision at the lower flow rates required in many applications. Theory. Under conditions of laminar (nonturbulent) flow, the flow rate of a fluid through a tube may be predicted by the Poiseuille equation: