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Separation of deoxyribonucleotides from ribonucleotides by paper chromatography
ANALYTICAL
Separation
22, 231-237 (1968)
BIOCHEMISTBY
of Deoxyribonucleotides by
Paper
BERNARD0 Eastern
Pennsylvania
Psychiatric
from
Ribonucleotides
Chromatography S. VANDERHEIDEN Institute,
Philadelphia,
Pennsylvania
1QlSQ
Received May 23, 1967
The methods available for the separation of deoxyribonucleotides from ribonucleotides are few and limited in scope so that their application is restricted to specific purposes. Thin layer chromatography has been used by Randerath (l-3) for rapid separation of very small amounts of nucleotides; paper chromatography in borate systems was used by Reichard (4), and Klenow and Lichtler (5)) but their data include only a few of the nucleotides. The same is true for methods based on ion-exchange column chromatography such as the ones described by Khym and Cohn (6) and Reichard (4). The availability of a simple method of preparation of SzP-labeled nucleotides (7) prompted the search for techniques suitable for the preparation of 32P-labeled deoxyribonucleotides free from the corresponding ribose analogs. In this report, paper chromatographic methods are described that make possible such preparations. MATERIALS
AND
METHODS
Deoxyribonucleotides (except d-UTP) , UTP, IMP, XMP, XDP, XTP, GMP, CMP, CDP, and CTP were obtained from Sigma, GTP, UDP, UMP, and d-UTP from Calbiochem, and IDP, ITP, GDP, AMP, ADP, and ATP from Pabst Laboratories. Acid-washed (1 N HCl followed by distilled water) Whatman No. 1 paper was used throughout. For use with solvent system III (ethanol/ borate) the acid-washed paper was dipped in O.lOM borate buffer, pH 10, and dried in air (8). Chromatography Solvent Systems
(I) Isobutyric acid/l N NH,OH/O.l M Na EDTA (pH 8.2) (50/30/ 0.8), used in descending chromatography, and developed from 18 to 48 hr depending on the nucleotides. (II) Absolute ethanol/l M ammonium acetate (pH 3.8)/l M Na 231
232
BERNARD0
S.
VANDERHEIDEN
EDTA (pH 8.2) (75/29/l) (9), used in descending chromatography and developed 24 to 48 hr. (III) 95% ethanol/O.lOM sodium borate buffer (pH 10) (60/49), used in ascending chromatography and developed 15 to 18 hr. METHODS
In most cases nucleotides were applied in the solid state by removing a small amount (estimated between 2 and 8 ,ug) with a capillary melting point tube (Kimax 34502)) placing it on the application point by tapping gently against the chromatography paper, and finally adding l-2 ~1 of distilled water to impregnate the paper. In some cases 2 ,ILI of a 50 pmole/ml solution was applied. Ascending chromatography was carried out using 11” X 16J’ acid-washed Whatman No. 1 paper, stapled to form cylinders 16”’ high. The sheets were allowed to equilibrate in individual 18” X 6” cylindric chromatography jars containing 100 ml of solvent, prior to development. Descending chromatography was carried out using 9’ X 21” sheets of acid-washed Whatman No. 1 paper. Rectangular chromatography jars 24@’X 125’ X 12’ were lined with filter paper saturated with an excess of the appropriate solvent. The chromatography sheets were allowed to equilibrate for 30 min prior to development. The compounds were detected by inspection under short-wave ultraviolet light. When orthophosphate was used as a reference marker the papers were dipped in molybdate reagent (10). RESULTS AND DISCUSSION
Figures l-3 illustrate the separation of ribo- and deoxyribonucleotides of analogous bases, and the phosphates of thymidine, inosine, and xanthosine in the solvent systems described. The corresponding Rf or Rp, values are listed in Table 1. Solvents I and II are suitable for the separation of the individual mono-, di-, and triphosphates of either deoxy- or ribonucleosides, while solvent III is satisfactory only for the deoxyribose derivatives. Solvent I provides good separation of the mono-, di-, and triphosphates of both series of compounds but, in spite of the fact that the deoxyribose compounds of a particular base have a slightly higher migration rate than the corresponding ribose analogs, some overlap exists between their corresponding phosphates. Best separation of the deoxy- and ribose derivatives of a particular base is obtained with solvent 1I.l ‘For a complex mixture of deoxy- and ribonucleotides the combination of highvoltage paper electrophoresis (7) and paper chromatography with one of the solvents described affords more complete separation (11).
FIG. 1. Paper chromatographic separation of adenine, cytosine, guanine, and uracil ribo- and deoxyribonucleotides, thymine deoxyribonucleotides, xanthine and hypoxanthine ribonucleotides. Approximately 2-8 pg of compound was applied on acid-washed Whatman No. 1 paper. Dark areas indicate the major compounds, full lines show UV-absorbing compounds, and broken lines, compounds containing phosphate. Solvent I was used in descending chromatography; adenine, cytosine, and thymine compounds were developed 24 hr, guanine, xanthine, and hypoxanthine compounds, 48 hr, and uracil compounds, 18 hr. 233
BERNARD0
S.
VANDEBHEIDEN
a 30-
205
lo-
08@
00
o
0 ,\:-: ,-. ,,
FIQ. 2. Paper chromatographic separation of adenine, cytosine, guanine, and uracil ribo- and deoxyribonucleotide, thymine deoxyribonucleotides, xanthine, and hypoxanthine ribonucleotides. (See figure 1.) Solvent II-descending chromatography; adenine, cytosine, uracil, and thymine compounds were developed 24 hr, gunnine, 42 br, and xanthine and hypoxanthine, 48 hr.
DEOXYRIBO-
AND
235
RIBONUCLEOTIDES
20
30
20
20
E 0
a0
0 00
10
0 00 0 iyi:
i
UMP lKP flp6uIpuDpauTp
Fm. 3. Paper chromatographic separation of ribo- and deoxyribonucleotides. Approximately 2-3 cg of compound was applied on acid-washed Whatman No. 1 previously dipped in 0.10M borate buffer, pH 10, and air-dried. Solvent III was used in ascending chromatography, developed 15-18 hr. Dark areas indicate the major compounds; W-absorbing impurities are shown in full lines. The use of ethanol/ammonium acetate mixtures for chromatography pyrimidine, and derivatives was described by Paladini and Leloir (12). Cain, Kushmerick, and Davies (9) found that addition of
of purine,
EDTA to the system increased the resolution for their particular separation. The same solvent was used more extensively by Vanderheiden and Boszormenyi-Nagy (7) for the separation of phosphate esters and nucleotides. The solvent described by Plesner (13) (ethanol/ammonium
236
BERNARD0
Rpi and Rt
Values
S.
VANDERHEXDEN
TABLE 1 of Deoxyribonucleotides
and Ribonucleotidesa
Solvent
so:vent Compound
I RP,
I$
III R/
Compound
d-AMP
1.58
0.93
0.44
AMP
1.36
0.75
0.17
d-ADP
1.33
0.46
0.32
ADP
1.11
0.34
0.11
d-ATP
1.18
0.26
0.23
ATP
0.94
0.19
0.07
d-CMP
1.45
0.92
0.46
CMP
1.21
0.68
0.17
0.96
0.29
0.09 0.06
d-CDP
1.13
0.45
0.33
CDP
d-CTP
0.79
0.23
0.23
CTP
0.77
0.16
d-GMP
1.19
0.68
0.29
GMP
0.86
0.54
0.08
d-GDP
0.91
0.31
0.20
GDP
0.64
0.24
0.06
d-GTP
0.74
0.15
0.13
GTP
0.50
0.11
0.04
d-UMP
1.07
1.12
0.46
UMP
0.83
0.86
0.17
d-UDP
0.77
0.55
0.34
UDP
0.62
0.38
0.10
d-UTP
0.60
0.26
0.24
UTP
0.50
0.19
0.06
d-TMP
1.27
1.17
0.48
IMP
0.79
0.68
0.15
d-TDP
0.94
0.69
0.38
IDP
0.54
0.28
0.10
d-TTP
0.76
0.41
0.28
ITP
0.39
0.15
0.07
Pi
1.00
1.00
0.26
0.09
RP* of different than
a The is
the nucleotides those shown
XMP
0.60
0.48
XDP
0.32
0.20
0.06
XTP
0.21
0.10
0.04
in solvents I and II may in Figures 1 and 2.
vary
if the time
of development
acetate saturated with borate) was used by Klenow and Lichtler (5) for the separation of deoxyribo- and ribonucleotides of adenine, and by Reichard (4) for the corresponding cytidine analogs. More recently Bieleski and Young (8) introduced mixtures of ethanol and borate buffer in the separation of phosphate esters from plant tissue. Although the migration constant (Rp,) for a large number of phosphate esters was given, the deoxyribonucleotides were not included. The proportions of their ethanol/borate buffer mixture was modified for better resolution using ascending chromatography and acid-washed Whatman No. 1 paper. Even though most samples were applied in the solid state in order to avoid possible breakdown occurring while the compounds are in solution, most of the di- and triphosphate standards showed variable amounts of impurities. Almost all of the triphosphates revealed the presence of a slower moving UV-absorbing phosphate compound, assumed to be the corresponding tetraphosphates (14). Some deoxyribonucleotide prepara-
DEOXYRIRO-
AND
RIBONUCLEOTIDES
237
‘tions showed the presence of the corresponding ribonucleotides, while the .xanthosine phosphates revealed the presence of compounds with migration rates similar to and suggestive of the inosine derivatives. The method described provides the means for checking the purity of *oommercial preparations when unequivocal interpretation of experimental *data is desired. In addition to identification, the method may be used -for the purification of small amounts of deoxy- and ribonucleotides. SUMMARY
Ribonucleotides of six, and deoxyribonucleotides of five, common purine .and pyrimidines were chromatographed on paper as 5’-mono-, di-, and triphosphates. One-dimensional separation was achieved between monoand polyphosphates or ribo- and deoxyribonucleotides of a given base or of certain pair of bases. The solvents used consisted of isobutyric/ ammonia, ethanol/ammonium acetate, or ethanol/borate mixtures. The method can be used for the purification or test of purity of small amounts of nucleotides. ACKNOWLEDGMENT The author is indebted to Mrs. Portia Russell for her technical assistance and to Miss Bertha Blackiston, a participant in the Summer Bio-Science Program for High School Students, who assisted in some of the technical aspects of this work. REFERENCES K., Biochim. Biophys. Acta 76, 622 (1963). K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). 3. NEUHARD, J., RANDERATW, E., AND RANDERATH, K., Anal. Biochem. 13, 211 (1965). 4. REICHABD, P., Acta Chem. Stand. 12, 2048 (1958). 5. KLENOW, H., AND LICHTLER, E., Biochim. Biophys. Acta 23,6 (1957). 6. KHYM, J. X., AND COHN, W. E., Biochim. Biophys. Acta 15, 139 (1954). 7. VANDERHEIDEN, B. S., AND BOSZORMENYI-NAGY, I., Anal. Biochem. 13, 496 (1965). S. BJELESKI, R. L., AND YOUNG, R. E., Anal. Biochem. 6, 54 (1963). 9. CAIN, D. F., KIJSHMERICK, M. J., AND DAVIES, R. E., Biochim. Biophys. Acta 74, 735 (1963). 10. Bmmows, S., GRYLLS, F. S. M., AND HARRISON, J. S., Nature 170, 800 (1952). 11. VANDEBHEIDEN, B. S., Anal. Biochem. 00,000 (196X). 12. PALADINI, A. C., AND LELOIR, L. F., Biochem. J. 51,426 (1952). 13. PLESNER, P., Acta Chem. Stand. 9, 197 (1955). 14. VANDEQHEIDEN, B. S., J. Biol. Chem. 241,3053 (1966). 1. RANDEBATH, 2. RANDERATH,
Separation
22, 231-237 (1968)
BIOCHEMISTBY
of Deoxyribonucleotides by
Paper
BERNARD0 Eastern
Pennsylvania
Psychiatric
from
Ribonucleotides
Chromatography S. VANDERHEIDEN Institute,
Philadelphia,
Pennsylvania
1QlSQ
Received May 23, 1967
The methods available for the separation of deoxyribonucleotides from ribonucleotides are few and limited in scope so that their application is restricted to specific purposes. Thin layer chromatography has been used by Randerath (l-3) for rapid separation of very small amounts of nucleotides; paper chromatography in borate systems was used by Reichard (4), and Klenow and Lichtler (5)) but their data include only a few of the nucleotides. The same is true for methods based on ion-exchange column chromatography such as the ones described by Khym and Cohn (6) and Reichard (4). The availability of a simple method of preparation of SzP-labeled nucleotides (7) prompted the search for techniques suitable for the preparation of 32P-labeled deoxyribonucleotides free from the corresponding ribose analogs. In this report, paper chromatographic methods are described that make possible such preparations. MATERIALS
AND
METHODS
Deoxyribonucleotides (except d-UTP) , UTP, IMP, XMP, XDP, XTP, GMP, CMP, CDP, and CTP were obtained from Sigma, GTP, UDP, UMP, and d-UTP from Calbiochem, and IDP, ITP, GDP, AMP, ADP, and ATP from Pabst Laboratories. Acid-washed (1 N HCl followed by distilled water) Whatman No. 1 paper was used throughout. For use with solvent system III (ethanol/ borate) the acid-washed paper was dipped in O.lOM borate buffer, pH 10, and dried in air (8). Chromatography Solvent Systems
(I) Isobutyric acid/l N NH,OH/O.l M Na EDTA (pH 8.2) (50/30/ 0.8), used in descending chromatography, and developed from 18 to 48 hr depending on the nucleotides. (II) Absolute ethanol/l M ammonium acetate (pH 3.8)/l M Na 231
232
BERNARD0
S.
VANDERHEIDEN
EDTA (pH 8.2) (75/29/l) (9), used in descending chromatography and developed 24 to 48 hr. (III) 95% ethanol/O.lOM sodium borate buffer (pH 10) (60/49), used in ascending chromatography and developed 15 to 18 hr. METHODS
In most cases nucleotides were applied in the solid state by removing a small amount (estimated between 2 and 8 ,ug) with a capillary melting point tube (Kimax 34502)) placing it on the application point by tapping gently against the chromatography paper, and finally adding l-2 ~1 of distilled water to impregnate the paper. In some cases 2 ,ILI of a 50 pmole/ml solution was applied. Ascending chromatography was carried out using 11” X 16J’ acid-washed Whatman No. 1 paper, stapled to form cylinders 16”’ high. The sheets were allowed to equilibrate in individual 18” X 6” cylindric chromatography jars containing 100 ml of solvent, prior to development. Descending chromatography was carried out using 9’ X 21” sheets of acid-washed Whatman No. 1 paper. Rectangular chromatography jars 24@’X 125’ X 12’ were lined with filter paper saturated with an excess of the appropriate solvent. The chromatography sheets were allowed to equilibrate for 30 min prior to development. The compounds were detected by inspection under short-wave ultraviolet light. When orthophosphate was used as a reference marker the papers were dipped in molybdate reagent (10). RESULTS AND DISCUSSION
Figures l-3 illustrate the separation of ribo- and deoxyribonucleotides of analogous bases, and the phosphates of thymidine, inosine, and xanthosine in the solvent systems described. The corresponding Rf or Rp, values are listed in Table 1. Solvents I and II are suitable for the separation of the individual mono-, di-, and triphosphates of either deoxy- or ribonucleosides, while solvent III is satisfactory only for the deoxyribose derivatives. Solvent I provides good separation of the mono-, di-, and triphosphates of both series of compounds but, in spite of the fact that the deoxyribose compounds of a particular base have a slightly higher migration rate than the corresponding ribose analogs, some overlap exists between their corresponding phosphates. Best separation of the deoxy- and ribose derivatives of a particular base is obtained with solvent 1I.l ‘For a complex mixture of deoxy- and ribonucleotides the combination of highvoltage paper electrophoresis (7) and paper chromatography with one of the solvents described affords more complete separation (11).
FIG. 1. Paper chromatographic separation of adenine, cytosine, guanine, and uracil ribo- and deoxyribonucleotides, thymine deoxyribonucleotides, xanthine and hypoxanthine ribonucleotides. Approximately 2-8 pg of compound was applied on acid-washed Whatman No. 1 paper. Dark areas indicate the major compounds, full lines show UV-absorbing compounds, and broken lines, compounds containing phosphate. Solvent I was used in descending chromatography; adenine, cytosine, and thymine compounds were developed 24 hr, guanine, xanthine, and hypoxanthine compounds, 48 hr, and uracil compounds, 18 hr. 233
BERNARD0
S.
VANDEBHEIDEN
a 30-
205
lo-
08@
00
o
0 ,\:-: ,-. ,,
FIQ. 2. Paper chromatographic separation of adenine, cytosine, guanine, and uracil ribo- and deoxyribonucleotide, thymine deoxyribonucleotides, xanthine, and hypoxanthine ribonucleotides. (See figure 1.) Solvent II-descending chromatography; adenine, cytosine, uracil, and thymine compounds were developed 24 hr, gunnine, 42 br, and xanthine and hypoxanthine, 48 hr.
DEOXYRIBO-
AND
235
RIBONUCLEOTIDES
20
30
20
20
E 0
a0
0 00
10
0 00 0 iyi:
i
UMP lKP flp6uIpuDpauTp
Fm. 3. Paper chromatographic separation of ribo- and deoxyribonucleotides. Approximately 2-3 cg of compound was applied on acid-washed Whatman No. 1 previously dipped in 0.10M borate buffer, pH 10, and air-dried. Solvent III was used in ascending chromatography, developed 15-18 hr. Dark areas indicate the major compounds; W-absorbing impurities are shown in full lines. The use of ethanol/ammonium acetate mixtures for chromatography pyrimidine, and derivatives was described by Paladini and Leloir (12). Cain, Kushmerick, and Davies (9) found that addition of
of purine,
EDTA to the system increased the resolution for their particular separation. The same solvent was used more extensively by Vanderheiden and Boszormenyi-Nagy (7) for the separation of phosphate esters and nucleotides. The solvent described by Plesner (13) (ethanol/ammonium
236
BERNARD0
Rpi and Rt
Values
S.
VANDERHEXDEN
TABLE 1 of Deoxyribonucleotides
and Ribonucleotidesa
Solvent
so:vent Compound
I RP,
I$
III R/
Compound
d-AMP
1.58
0.93
0.44
AMP
1.36
0.75
0.17
d-ADP
1.33
0.46
0.32
ADP
1.11
0.34
0.11
d-ATP
1.18
0.26
0.23
ATP
0.94
0.19
0.07
d-CMP
1.45
0.92
0.46
CMP
1.21
0.68
0.17
0.96
0.29
0.09 0.06
d-CDP
1.13
0.45
0.33
CDP
d-CTP
0.79
0.23
0.23
CTP
0.77
0.16
d-GMP
1.19
0.68
0.29
GMP
0.86
0.54
0.08
d-GDP
0.91
0.31
0.20
GDP
0.64
0.24
0.06
d-GTP
0.74
0.15
0.13
GTP
0.50
0.11
0.04
d-UMP
1.07
1.12
0.46
UMP
0.83
0.86
0.17
d-UDP
0.77
0.55
0.34
UDP
0.62
0.38
0.10
d-UTP
0.60
0.26
0.24
UTP
0.50
0.19
0.06
d-TMP
1.27
1.17
0.48
IMP
0.79
0.68
0.15
d-TDP
0.94
0.69
0.38
IDP
0.54
0.28
0.10
d-TTP
0.76
0.41
0.28
ITP
0.39
0.15
0.07
Pi
1.00
1.00
0.26
0.09
RP* of different than
a The is
the nucleotides those shown
XMP
0.60
0.48
XDP
0.32
0.20
0.06
XTP
0.21
0.10
0.04
in solvents I and II may in Figures 1 and 2.
vary
if the time
of development
acetate saturated with borate) was used by Klenow and Lichtler (5) for the separation of deoxyribo- and ribonucleotides of adenine, and by Reichard (4) for the corresponding cytidine analogs. More recently Bieleski and Young (8) introduced mixtures of ethanol and borate buffer in the separation of phosphate esters from plant tissue. Although the migration constant (Rp,) for a large number of phosphate esters was given, the deoxyribonucleotides were not included. The proportions of their ethanol/borate buffer mixture was modified for better resolution using ascending chromatography and acid-washed Whatman No. 1 paper. Even though most samples were applied in the solid state in order to avoid possible breakdown occurring while the compounds are in solution, most of the di- and triphosphate standards showed variable amounts of impurities. Almost all of the triphosphates revealed the presence of a slower moving UV-absorbing phosphate compound, assumed to be the corresponding tetraphosphates (14). Some deoxyribonucleotide prepara-
DEOXYRIRO-
AND
RIBONUCLEOTIDES
237
‘tions showed the presence of the corresponding ribonucleotides, while the .xanthosine phosphates revealed the presence of compounds with migration rates similar to and suggestive of the inosine derivatives. The method described provides the means for checking the purity of *oommercial preparations when unequivocal interpretation of experimental *data is desired. In addition to identification, the method may be used -for the purification of small amounts of deoxy- and ribonucleotides. SUMMARY
Ribonucleotides of six, and deoxyribonucleotides of five, common purine .and pyrimidines were chromatographed on paper as 5’-mono-, di-, and triphosphates. One-dimensional separation was achieved between monoand polyphosphates or ribo- and deoxyribonucleotides of a given base or of certain pair of bases. The solvents used consisted of isobutyric/ ammonia, ethanol/ammonium acetate, or ethanol/borate mixtures. The method can be used for the purification or test of purity of small amounts of nucleotides. ACKNOWLEDGMENT The author is indebted to Mrs. Portia Russell for her technical assistance and to Miss Bertha Blackiston, a participant in the Summer Bio-Science Program for High School Students, who assisted in some of the technical aspects of this work. REFERENCES K., Biochim. Biophys. Acta 76, 622 (1963). K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). 3. NEUHARD, J., RANDERATW, E., AND RANDERATH, K., Anal. Biochem. 13, 211 (1965). 4. REICHABD, P., Acta Chem. Stand. 12, 2048 (1958). 5. KLENOW, H., AND LICHTLER, E., Biochim. Biophys. Acta 23,6 (1957). 6. KHYM, J. X., AND COHN, W. E., Biochim. Biophys. Acta 15, 139 (1954). 7. VANDERHEIDEN, B. S., AND BOSZORMENYI-NAGY, I., Anal. Biochem. 13, 496 (1965). S. BJELESKI, R. L., AND YOUNG, R. E., Anal. Biochem. 6, 54 (1963). 9. CAIN, D. F., KIJSHMERICK, M. J., AND DAVIES, R. E., Biochim. Biophys. Acta 74, 735 (1963). 10. Bmmows, S., GRYLLS, F. S. M., AND HARRISON, J. S., Nature 170, 800 (1952). 11. VANDEBHEIDEN, B. S., Anal. Biochem. 00,000 (196X). 12. PALADINI, A. C., AND LELOIR, L. F., Biochem. J. 51,426 (1952). 13. PLESNER, P., Acta Chem. Stand. 9, 197 (1955). 14. VANDEQHEIDEN, B. S., J. Biol. Chem. 241,3053 (1966). 1. RANDEBATH, 2. RANDERATH,