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The value of Dowex-50 in fractionation of nucleotides from acid soluble pool. A caution
ANALYTICAL
BIOCHEMISTRY
The Value
(1974)
of Dowex-50 in Fractionation of Nucleotides from Acid Soluble Pool. A Caution
MIROSLAV Department
61, 568-512
BATES
of Physiology,
AND
NEBOJSA
Faculty
of Medicine, Yugoslavia
Zagreb, Received
September
28, 1973;
accepted
AVDALOVIC University
April
of
Zagreb,
29, 1974
Problems were encountered in separating acid soluble nucleotides into four main groups by chromatography on DowexSO(H+) columns. These difficulties derived from the low affinity of ATP, ADP, GDP, GTP, CDP, and CTP for Dowex-56 (H’) ion exchanger under mild acid conditions. Thus, under the conditions described, these compounds travel together with uridine compounds.
The method of Katz and Comb (1) is in widespread use for separation of nucleotide monophosphates after alkaline hydrolysis of RNA. According to Kochakian and Hill (2) this method can be also used to separate acid soluble nucleotides into four main groups. However, they obtained an exceedingly high value for UTP concentration in the mouse kidney. Further, after an in viva injection of labeled erotic acid a rather poor incorporation of radioactivity into UTP was also recorded. In this communication we present data that do not support the use of the Katz and Comb (1) procedure for the group separation of nucleotides. We also put forward an explanation for the “anomalous” behaviour of UTP labeling after an injection of radioactive erotic acid. MATERIALS
AND
METHODS
The acid insoluble residue obtained after standard perchloric acid precipitation of the total mouse kidney homogenate was washed once with 0.3 N perchloric acid, twice with methanol, and then incubated in 0.3 N KOH at 37°C for 16-20 hr. Yeast RNA (British Drug Houses) was directly dissolved in distilled water, and an equal volume of 0.6 N KOH was added. The samples were then incubated as described above. After incubation, a double volume of 0.6 N perchloric acid was added, the samples were left for 10 min in the cold and the resulting precipitate was removed by centrifugation. A portion of the clear supernatant Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
508 Inc. reserved.
DOWEX-50
CHROMATOGRAPHY
509
solution was adsorbed on activated charcoal (Merck). After a thorough washing with water, the nucleotides were desorbed from charcoal with an ammonia-ethanol-water mixture according to Tsuboi and Price (3). The eluate was evaporated to dryness in a Biichi rotatory evaporator. The dry residue was dissolved either in distilled water or in 0.05 or 0.01 N HCI. Ion-exchange chromatography of the RNA hydrolysate as well as of the acid soluble nucleot.ides was performed exactly according to Katz and Comb (1) and Kochakian and Hill (2). The acid soluble fraction was prepared according to Kochakian and Hill (2) or Bucher and Swaffield (4j, and thereafter subjected t.o charcoal adsorption, elution and concentration as described above for the RNA hydrolysate. Thin-Layer
Chromatography
In this study PEI-f cellulose precoated glass plates (Merck) were used. Standard samples of pure nucleotides (Boehringer Mannheim, GmbH) or desalted and concentrated samples from a Dowex-50 (H’) column were applied as a spot or streaked on 20 X 20 cm thin-layer plates. The plates were developed at room temperature (23-25°C). Separation of ilTP, ADP, and AMP was achieved according to Randerath and Randerath (5,6), as modified by Bucher and Swaffield (4). Nucleot,ides were detected by examining the plates in ultraviolet light (CAMG uv lamp, Switzerland). Quantitative elution of nucleotides from the thin-layer plates was done according to Randerath and Randerath (5). The eluted samples were diluted with 0.01 N HCI and absorbances were det,ermined against appropriate blanks at 254 nm in a Zeiss VSU-1 spectrophotometer. RESULTS
AND DISCUSSION
The chromatographic patterns of the total mouse kidney and yeast RNA alkaline hydrolysate were identical to that described by Katz and Comb il). Figure 1 shows the behaviour of the acid soluble fraction from the mouse kidney homogenate on the Dowex-50(H+) column, When the acid soluble pool was prepared with liquid nitrogen following all precautions suggested by Bucher and Swaffield (4), the first peak was much higher than in the samples prepared according to Kochakian and Hill (2). The fractions under the major peaks (I, II, and III + IV) were pooled and adsorbed on charcoal and then processed as described above for thin-layer chromatography. The combined samples from the first peak revealed the presence of tri- and diphosphates of A and G group and also IJMP and UDPG. Among the tri- and diphosphates SO-90% belonged to the A group compounds. The precise quantitation of the
510
BATES
AND
AVDALOVI6
.i
2.0
I:dl
FIG. 1. Ion-exchange chromatography on DowexZiO(H+). Elution pattern of the acid soluble nucleotides prepared with (0-O) and without (a-1 liquid nitrogen. Fractions under the peaks I, II, and (III + IV) were adsorbed on charcoal, eluted and concentrated as described in the text., and then subjected to thin-layer chromatography on PEI-f cellulose. The first or “U” peak besides uridine nucleotides contained adenine and guanine nucleotides as determined by the enzymatic procedure (11). Second or “G” peak contained GMP and also flavin nucleotides. Combined III + IV peak contained mostly AMP and some unidentified compound. The same experiment was performed on the rat kidney and the guinea pig kidney acid soluble nucleotides. The described elution pattern was uniformly recorded in all nine experiments.
separated nucleotides was not attempted. Since the first peak (2) should have contained U compounds only, we decided to chromatograph samples of pure nucleotides on Dowex-50(H+) columns, Chromatography was carried out under the same conditions as described for the acid soluble pool (2). It is obvious from Fig. 2 that ATP and ADP move together with U compounds in the first peak. Thus, in spite of the acid conditions, the presence of the secondary and tertiary phosphate groups profoundly influenced the expression of the cationic properties of the purine compounds. To remove adenine or adenosine from the same column it was necessary to use much stronger acid (2 N HCl). Some theoretical aspects and the possible basis for such a behaviour can be found in the articles of Elving (7) and Kennard et al. (8). If such a heterogenous mixture from the first or “U” peak were further fractionated on Dowex-1 (9) as Kochakian and Hill (2) have done, one realizes (a) why the authors obtained exceedingly high values for t.he UTP content of the mouse kidney and (b) why there was an “anomalous” behaviour of UTP labeling after an injection of radioactive
DOWEX-50
511
CHROMATOGRAPHY
FRncTioNs(ml) FIG. 2. Ion-exchange chromatography of pure nucleotides on DowexSO(H+) column ~200400 mesh). The elution pattern of all tested nucleotides was not substantially changed by eluting with 0.01 N HCl instead of 0.05 N HCl. ATP ADP; FMN; (m-m) ~/AMP.
(O---O); (a-0)
(v--v) UMP; (v-v)
acid. The reason is that substantial amounts of ATP were present in the UTP fraction. It was also noticed (2) that the second or “G” peak did not give the characteristic spectrophotometric absorption curve. The same result was obtained in our experiments but we also found that this “G” fraction contained also flavin nucleotides. It is possible that the presence of flavin nucleotides masked the characteristic curve of the “G” compound(s) . Flavin nucleotides were characterized and identified according to Bessey et al. (10).
erotic
ACKNOWLEDGMENT This work was supported Council of Croatia.
in part. by Grants provided
by the Scientific
Research
REFERENCES
S..I\ND
1. K.4~2, 2. KOCHAKIAN,
3. TSU~I, 4. BUCHER,
COMB,
C. D., K. K., AND N. L. R.,
D. G. (1963) J. BioE. Ckem. 238, 3065. J. (1966) Biochemistry 5, 1696. D. (1959) Arch. Biochem. Biophys. 81, 223. AND SWAFFIELD, M. N. (1968) Biochim. Biophys. Acta 174, AND HILL, PRICE, T.
491. K., AND RANDERATH, E. (1968) in Methods in Enzymology part $ (L. Grossman and K. Moldave, eds.), Vol. 12. p. 323, Academic Press, New York, London.
5. RANDERATH,
512
BATES
AND
AVDALOVI6
6. RANDERATH, E., AND RANDERATH, K. (1965) Anal. B&hem. 12, 83. 7. ELVING, P. (1969) Electronic aspects of biochemistry, Ann. N. Y. Acad. Sci. 158, 124. 8. KENNARD, O., ISAACS, N. W., MOTHERWELL, W. D. S., COPPOLA, J. C., WAMPLER, D. L., LARSON, A. C., AND WATSON, D. G. (1971) Proc. Roy. Sot. London A 325, 401. 9. KHYM, .J. X., AND COHN, W. E. (19531 1. Amer. Chem. Sot. 75, 1153. 10. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H. (1949) J. Biol. Chem. 180, 755. 11. LAMPRECHT, W., UND TRAUTSCHOLD, I. (1970) in H. U. Bergmeyer, Methoden der enzymatischen Analyse, 2 Auflage, Band II, 2024 Verlag Chemie, Weinheim/Bergst.
BIOCHEMISTRY
The Value
(1974)
of Dowex-50 in Fractionation of Nucleotides from Acid Soluble Pool. A Caution
MIROSLAV Department
61, 568-512
BATES
of Physiology,
AND
NEBOJSA
Faculty
of Medicine, Yugoslavia
Zagreb, Received
September
28, 1973;
accepted
AVDALOVIC University
April
of
Zagreb,
29, 1974
Problems were encountered in separating acid soluble nucleotides into four main groups by chromatography on DowexSO(H+) columns. These difficulties derived from the low affinity of ATP, ADP, GDP, GTP, CDP, and CTP for Dowex-56 (H’) ion exchanger under mild acid conditions. Thus, under the conditions described, these compounds travel together with uridine compounds.
The method of Katz and Comb (1) is in widespread use for separation of nucleotide monophosphates after alkaline hydrolysis of RNA. According to Kochakian and Hill (2) this method can be also used to separate acid soluble nucleotides into four main groups. However, they obtained an exceedingly high value for UTP concentration in the mouse kidney. Further, after an in viva injection of labeled erotic acid a rather poor incorporation of radioactivity into UTP was also recorded. In this communication we present data that do not support the use of the Katz and Comb (1) procedure for the group separation of nucleotides. We also put forward an explanation for the “anomalous” behaviour of UTP labeling after an injection of radioactive erotic acid. MATERIALS
AND
METHODS
The acid insoluble residue obtained after standard perchloric acid precipitation of the total mouse kidney homogenate was washed once with 0.3 N perchloric acid, twice with methanol, and then incubated in 0.3 N KOH at 37°C for 16-20 hr. Yeast RNA (British Drug Houses) was directly dissolved in distilled water, and an equal volume of 0.6 N KOH was added. The samples were then incubated as described above. After incubation, a double volume of 0.6 N perchloric acid was added, the samples were left for 10 min in the cold and the resulting precipitate was removed by centrifugation. A portion of the clear supernatant Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
508 Inc. reserved.
DOWEX-50
CHROMATOGRAPHY
509
solution was adsorbed on activated charcoal (Merck). After a thorough washing with water, the nucleotides were desorbed from charcoal with an ammonia-ethanol-water mixture according to Tsuboi and Price (3). The eluate was evaporated to dryness in a Biichi rotatory evaporator. The dry residue was dissolved either in distilled water or in 0.05 or 0.01 N HCI. Ion-exchange chromatography of the RNA hydrolysate as well as of the acid soluble nucleot.ides was performed exactly according to Katz and Comb (1) and Kochakian and Hill (2). The acid soluble fraction was prepared according to Kochakian and Hill (2) or Bucher and Swaffield (4j, and thereafter subjected t.o charcoal adsorption, elution and concentration as described above for the RNA hydrolysate. Thin-Layer
Chromatography
In this study PEI-f cellulose precoated glass plates (Merck) were used. Standard samples of pure nucleotides (Boehringer Mannheim, GmbH) or desalted and concentrated samples from a Dowex-50 (H’) column were applied as a spot or streaked on 20 X 20 cm thin-layer plates. The plates were developed at room temperature (23-25°C). Separation of ilTP, ADP, and AMP was achieved according to Randerath and Randerath (5,6), as modified by Bucher and Swaffield (4). Nucleot,ides were detected by examining the plates in ultraviolet light (CAMG uv lamp, Switzerland). Quantitative elution of nucleotides from the thin-layer plates was done according to Randerath and Randerath (5). The eluted samples were diluted with 0.01 N HCI and absorbances were det,ermined against appropriate blanks at 254 nm in a Zeiss VSU-1 spectrophotometer. RESULTS
AND DISCUSSION
The chromatographic patterns of the total mouse kidney and yeast RNA alkaline hydrolysate were identical to that described by Katz and Comb il). Figure 1 shows the behaviour of the acid soluble fraction from the mouse kidney homogenate on the Dowex-50(H+) column, When the acid soluble pool was prepared with liquid nitrogen following all precautions suggested by Bucher and Swaffield (4), the first peak was much higher than in the samples prepared according to Kochakian and Hill (2). The fractions under the major peaks (I, II, and III + IV) were pooled and adsorbed on charcoal and then processed as described above for thin-layer chromatography. The combined samples from the first peak revealed the presence of tri- and diphosphates of A and G group and also IJMP and UDPG. Among the tri- and diphosphates SO-90% belonged to the A group compounds. The precise quantitation of the
510
BATES
AND
AVDALOVI6
.i
2.0
I:dl
FIG. 1. Ion-exchange chromatography on DowexZiO(H+). Elution pattern of the acid soluble nucleotides prepared with (0-O) and without (a-1 liquid nitrogen. Fractions under the peaks I, II, and (III + IV) were adsorbed on charcoal, eluted and concentrated as described in the text., and then subjected to thin-layer chromatography on PEI-f cellulose. The first or “U” peak besides uridine nucleotides contained adenine and guanine nucleotides as determined by the enzymatic procedure (11). Second or “G” peak contained GMP and also flavin nucleotides. Combined III + IV peak contained mostly AMP and some unidentified compound. The same experiment was performed on the rat kidney and the guinea pig kidney acid soluble nucleotides. The described elution pattern was uniformly recorded in all nine experiments.
separated nucleotides was not attempted. Since the first peak (2) should have contained U compounds only, we decided to chromatograph samples of pure nucleotides on Dowex-50(H+) columns, Chromatography was carried out under the same conditions as described for the acid soluble pool (2). It is obvious from Fig. 2 that ATP and ADP move together with U compounds in the first peak. Thus, in spite of the acid conditions, the presence of the secondary and tertiary phosphate groups profoundly influenced the expression of the cationic properties of the purine compounds. To remove adenine or adenosine from the same column it was necessary to use much stronger acid (2 N HCl). Some theoretical aspects and the possible basis for such a behaviour can be found in the articles of Elving (7) and Kennard et al. (8). If such a heterogenous mixture from the first or “U” peak were further fractionated on Dowex-1 (9) as Kochakian and Hill (2) have done, one realizes (a) why the authors obtained exceedingly high values for t.he UTP content of the mouse kidney and (b) why there was an “anomalous” behaviour of UTP labeling after an injection of radioactive
DOWEX-50
511
CHROMATOGRAPHY
FRncTioNs(ml) FIG. 2. Ion-exchange chromatography of pure nucleotides on DowexSO(H+) column ~200400 mesh). The elution pattern of all tested nucleotides was not substantially changed by eluting with 0.01 N HCl instead of 0.05 N HCl. ATP ADP; FMN; (m-m) ~/AMP.
(O---O); (a-0)
(v--v) UMP; (v-v)
acid. The reason is that substantial amounts of ATP were present in the UTP fraction. It was also noticed (2) that the second or “G” peak did not give the characteristic spectrophotometric absorption curve. The same result was obtained in our experiments but we also found that this “G” fraction contained also flavin nucleotides. It is possible that the presence of flavin nucleotides masked the characteristic curve of the “G” compound(s) . Flavin nucleotides were characterized and identified according to Bessey et al. (10).
erotic
ACKNOWLEDGMENT This work was supported Council of Croatia.
in part. by Grants provided
by the Scientific
Research
REFERENCES
S..I\ND
1. K.4~2, 2. KOCHAKIAN,
3. TSU~I, 4. BUCHER,
COMB,
C. D., K. K., AND N. L. R.,
D. G. (1963) J. BioE. Ckem. 238, 3065. J. (1966) Biochemistry 5, 1696. D. (1959) Arch. Biochem. Biophys. 81, 223. AND SWAFFIELD, M. N. (1968) Biochim. Biophys. Acta 174, AND HILL, PRICE, T.
491. K., AND RANDERATH, E. (1968) in Methods in Enzymology part $ (L. Grossman and K. Moldave, eds.), Vol. 12. p. 323, Academic Press, New York, London.
5. RANDERATH,
512
BATES
AND
AVDALOVI6
6. RANDERATH, E., AND RANDERATH, K. (1965) Anal. B&hem. 12, 83. 7. ELVING, P. (1969) Electronic aspects of biochemistry, Ann. N. Y. Acad. Sci. 158, 124. 8. KENNARD, O., ISAACS, N. W., MOTHERWELL, W. D. S., COPPOLA, J. C., WAMPLER, D. L., LARSON, A. C., AND WATSON, D. G. (1971) Proc. Roy. Sot. London A 325, 401. 9. KHYM, .J. X., AND COHN, W. E. (19531 1. Amer. Chem. Sot. 75, 1153. 10. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H. (1949) J. Biol. Chem. 180, 755. 11. LAMPRECHT, W., UND TRAUTSCHOLD, I. (1970) in H. U. Bergmeyer, Methoden der enzymatischen Analyse, 2 Auflage, Band II, 2024 Verlag Chemie, Weinheim/Bergst.