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Chromatographic separation and identification of nucleotides from animal tissues and yeast cells on a polyethyleneimine cellulose column
ANALYTICAL
BIOCHEMISTRY
81,
136- 142 (1977)
Chromatographic Separation and Identification of Nucleotides from Animal Tissues and Yeast Cells on a Polyethyleneimine Cellulose Column K.-H. lnstittrrr
fiir
Pharntahologir Laltnberge
urrd 355,
PFL~~GER
Tu.rikologie MarbtrrglLabn.
der
Pltilipps-Uttir,ersitrit West
Marburg.
Germany
Received July 13, 1976: accepted March 28. 1977 A procedure has been developed for the separation and identification of nucleotides by anion-exchange chromatography. The method includes column chromatography on PEI-cellulose with an unbuffered exponential salt gradient. This easily handled procedure gives a high degree of resolution and reproducibility and demands no personal attendance during elution. Application of the method is demonstrated with a mouse liver extract and extracts of yeast cells (Saccltarowtyces
cere~‘i.viae).
For separation of nucleotide mixtures obtained from different tissues by acid extraction, a great number of procedures have been developed employing chromatographic techniques on paper (l-6), on thin-layer plates (7-ll), and on columns (12-31), using numerous materials like Dowex (12-15), Sephadex (16,17), ECTEOLA-cellulose (18,19), TEAEcellulose (20), DEAE-cellulose (21-231, PEI-cellulose (23,24), PVP (25, 26), and cation exchangers (27,28). Some of these procedures are very suitable for special problems, such as the purification of nucleotides (25, 26) or the separation of mixtures containing a small number of compounds (15- 17.25-28). Unfortunately, all these techniques revealed several disadvantages when complex mixtures of soluble tissue nucleotides were used and when as many possible individual constituents of the mixture were to be resolved in one run. Chromatographic techniques on thin-layer plates are rapid and relatively easy to carry out. Even with complicated mixtures, they give good separation (7- 11). They are, however, unsuitable for a comprehensive quantitative analysis of unlabeled tissue extracts because of the high salt concentration and the variation in the composition of these extracts. Because of its greater sample capacity, column chromatography is far better suited for such problems, in particular for those conditions in which the largest possible number of individual constituents is to be measured. The techniques of column chromatography described so far are carried out 136 ISSN 0003.2697
NUCLEOTIDE
SEPARATION
ON
A PEI COLUMN
137
with 1300-6000 ml of acidic elution buffer and 30-60 hr of separation time (I?- 15, 22-24). Under these conditions, certain acid-labile materials can be destroyed. Furthermore, as many as five different concentration gradients are used for the separation process (12- 15.22). thereby requiring the presence of supervisory personnel. Cumbersome and costly high-pressure liquid column chromatography requires only 1-6 hr (29-31) for a separation, but uses polystyrene ion exchangers. This does not give good separation when tissue extracts are analyzed, as phenolic substances cause nonionic interactions with these resins. In view of these problems. a column chromatographic method was developed which makes possible a quantitative and reproducible resolution of acid-soluble unpurified tissue extracts, using only one neutral unbuffered NaCl or LiCl concentration gradient. PEI-cellulose was selected as an anion exchanger, as this material gives by far the best separation for nucleotide mixtures on thin-layer plates (9- 11). The method described here uses concave gradients, achieves a high sensitivity to detection using an elution volume of only 300 ml, and separates tissue extracts into as many as 25 symmetrical peaks in 16-20 hr. This method thus makes possible a comprehensive quantitative analysis of individual constituents, without the presence of supervisory personnel during the separation process. Furthermore, this method is less susceptible to complications and requires a very simple preparation of the column. METHODS C’hemicrrls. Nucleotides, nucleosides, nucleobases, UDP-glucose. ADP- and UDP-glucuronate, ApA, NAD, NADP, riboflavin 5phosphate, coenzyme A, folic acid, and riboflavin were purchased from Boehringer, Mannheim, W. Germany. [6-3H]uracil and [Ylinosin were obtained from Amersham Buchler, Braunschweig, W. Germany. OMP was from CalBiochem, Luzern, Switzerland. Acetylcoenzyme A and the mixture of oligonucleotides were prepared in our laboratory by methods described by Simon and Shemin (32) and Shapiro (33), respectively. PEI-Cellulose and the other chemicals were obtained from E. Merck, Darmstadt, W. Germany. All solutions were prepared with deionized glass-distilled water. Prepur-ation of the ion e.uchangc~r. PEI-Cellulose, 50.0 g, was vigorously stirred in 200 ml of 1 M NaCl. Then, the cellulose was allowed to sediment. and the yellowish overlay was siphoned off. In this manner, the washing was repeated four or five times. The sediment was then suspended in about 100 ml of 1 M NaCl and was carefully removed. This slurry is suitable for filling the columns, but can also be stored, well sealed, in the dark for up to 4 weeks without any loss of quality.
138
K.-H.
PFLLIGER
The column is made of cylindrical glass tubing (0.9 x 25 cm) which is tapered off at both ends. The outlet is connected with a thin silicone tube. It has a capacity of about 7.0 g of PEI-cellulose (dry weight), adsorbing a total of 1.5 mol of ATP. The top of the column is connected to a funnel which allows a hydrostatic pressure of 40 cm. The valve is closed, and the tapering outlet is plugged with a wet pea-sized piece of glass wool. The column is filled with evacuated I M NaCl, and the prepared ion exchanger is poured into the funnel. After several hours of settling, the valve is opened, and the column is allowed to drip with a hydrostatic pressure of 40 cm for 4 hr. Now, the excess ion exchanger is carefully Preparation
of the column.
Tronsmlttance 4 30
Molortty
at 260 nm
of NaCl
PH 8.0
LO.
6.0
50
L.0.
1
30 2.0. 1.0. L
gradlent
6
6
IO
12
11
16
16
20
22 Tune lhl
elution
Absorbance 1‘ 1.2 1.0
i
&gin of gradlent elurlo” , AMP
,ADP
ATP
0.6. 0.6.
FIG. 1. (a) Chromatography of an extract of 250 mg (wet weight) of yeast cells (Saccharomyces cerevisiue) previously grown in Williamson and Scopes (34) medium. The yeast was harvested on a membrane filter, washed with ice-cold water. and extracted for IS was neutralized with potassium min with ice-cold 0.6 M perchloric acid at 2°C. The extract hydroxide and was centrifuged to remove the cell residue and the potassium perchlorate. The neutral extract was used immediately for chromatography or was lyophilized for storage. For the numbers of the peaks, see Table 1. (b) Chromatography ofa mouse liver extract. The mice were killed. and the livers were rapidly excised with pliers precooled in liquid nitrogen. The liver was completely frozen in nitrogen and was shattered in a steel mortar. The extraction and the following preparation were similar to the procedure described for yeast cells, except that the cell residue was removed before neutralization.
NUCLEOTIDE
SEPARATION
ON
A PEI COLUMN
139
siphoned off, the funnel is cleaned, and the column is equilibrated with glass-distilled water for at least 5 hr. The column is then ready for use. It is possible to regenerate the columns after operation by washing them for 4 hr with water. Thus it is possible to use them for as many as five separations. Unfortunately, the resistance to Bow increases with each operation of the column. Usually, the columns were used for only one analysis. Starzdard elution system. At the beginning, the mixing chamber contains 240 ml of glass-distilled water. NaCl, 4.15 M was pumped into the mixing chamber at a flow rate of 4.0 mlihr, while the elution flow was maintained at 16.0 ml/hr by a metering pump (Fa. Biihler. Tiibingen, W. Germany). The duration of gradient elution was 18-20 hrs: the elution volume was about 300 ml. Ehtion arzd identi$cation of eluted materials. For initial chromatographic studies, nucleotide mixtures composed of lo- 16 compounds (100 pg of each nucleotide) are applied to the columns in 2-3 ml of water. The neutralized extracts from 150-2.50 mg (wet weight) of animal tissues or yeast cells are dissolved in 5- 10 ml of water. After absorption of the solutions, the column is washed for 2-3 hr with water at a flow rate of 16 ml/hr to remove ribonucleosides, bases, and other nonanionic compounds. Now. the columns are eluted with the exponential salt gradient. The eluates are monitored at 257 or 260 and 280 nm synchronously, and the collected nucleotide materials in each elution peak were identified on the basis of the following criteria. (i) Ultraviolet absorption spectra of the pooled peaks at ph 2.0, 7.0, and 12.0 were determined on a Zeiss DMR 21 spectrophotometer: (ii) coincident elution from the column with authentic samples added to the tissue extracts: (iii) The elution patterns of radioactively labeled extracts by incorporation of [6-“Hluracil or [‘“Clinosine into living cells. RESULTS
Under the described condition. following equation:
AND DISCUSSION
the exponential L.,n , -“A A -1
salt gradient yields the
L’ IIs, I.
where t (min) = time; A (ml. min-‘) = operation flow rate; Z (ml. min-I) = flow rate of salt solution pumped into the mixing chamber: M, (ml) = volume of water in the mixing chamber at the point of time t = 0: K, (mol. 1-l) = concentration of the added salt solution; K, (mol. l-‘) = salt concentration of the gradient at point of time t.
140
K.-H. Absorbance
PFLOGER
ot 260 “In
@agin of gradlent
elutnn
FIG. 2. Chromatography of an extract ously exposed to [3H]uridine (0.1 PCiiml) were extracted by the usual procedure.
Volume of
eluate
Cm!1
of yeast cells (Succhcrvonzyceu rere~Giae) previin Williamson and Scopes (34) medium. The cells
In this paper are specified the data of the standard gradient which was found most acceptable for the resolution of tissue extracts. The length, the slope, and the final height of the gradient can be varied for special separation problems by varying M,,, and/or K,, and/orZ, and/or A.
The results of chromatographic separation of extracts from yeast cells and liver tissue, using the standard gradient, are shown in Figs. la, b and 2. Table 1 presents the retention times and the localization of more than 40 standard substances by coincident elution from the column with yeast-cell extracts. The high degree of resolution and reproducibility has been already documented by more than 400 chromatograms of complex mixtures such as tissue extracts. Rustum and Schwartz (23) have described a useful separation method for acid-soluble tissue extracts on DEAE-cellulose. In a comparison of this matrix with PEI-cellulose, they found that the latter could not be stored as easily and showed a considerable batch variability, thus giving only incomplete separations. These disadvantages of PEI-cellulose were overcome by using the simple method for preparation of the commercial material and a standard column preparation method, with the result that PEI-cellulose, in all respects, proved to be significantly superior to DEAE-cellulose in work with neutral eluents. The standard nucleotides like mono-, di-, and triphosphates were recovered in a yield of 92-101%. Only ADP gave a yield of 78%. The fractions are eluted in volumes of 5-15 ml involving a high sensitivity to detection and a relatively small contamination by salt. When NaCl is replaced by LiCl, the salt can be removed by ethanol extraction. When the
NUCLEOTIDE
SEPARATION TABLE
RLTENTION
Retention thr)
with
Gradient 1.97 1.13 2.43 2.9 3.3 3.6 3.83 4.43 4.76
elution
5.83 6.3 6.5 6.43 7.43 7.63 8.8 9.9 10.7 II.0 II.5 17.13 17.9 13.7 14.2 14.6 15.2 16.8
141
A PEI COLUMN
I
TIMES DFTHE CohlPouNos OF YEAST AND STANDARD SURSl'ANCTS
time
Washing O-2.0
ON
Localization of standard substances
EXTRACTS
No. of elution peak
H,O Bases. nucleosides, acids. riboflavin. pyridoxine
Orotic NAD, CAMP
acid ApA,
amino aneurine.
oligonucleotides
I la ?
UDP-Glucose ADP-Glucose UMP. dTMP CMP. dCMP 5’-AMP. 3’-AMP. 2’.AMP. dAMP GMP, dGMP (II Maximum of riboflavin-PO, peak (2) Maximum of riboflavin-PO, peak UDP-Glucuronate NADP OMP. folic acid UDP CDP ADP GDP Acetyl-CoA UTP, dTTP CTP. ITP ATP CoA-SH GTP Adenosine tetraphosphate, RNA fractions
3 4 4a 5 6 I 8 8a Xa 9 IO II I:! I3 I4 I5 I6 l6a I7 l7a I8 I9
pH of the eluate is maintained in the range of pH 6.5-7.0. acid-labile materials are hardly destroyed during chromatography. Only during the elution of acidic material like amino acids, folic acid, etc., do values as low as pH 4.0 occur. This simple and practical procedure offers a number
142
K.-H.
PFLiiGER
of advantages over existing methods and should permit further studies of the nucleotide metabolism. ACKNOWLEDGMENTS For calculation of the equation. the author would like to thank Mr. F. J. Thome, Institut fur medizinische und biologische Statistik Marburg, W. Germany.
Mr. G. Albricht and und Dokumentation.
REFERENCES 1. Kunz, W., Schmid. W.. and Siess. M. (1958) Z. Gesamfe Exp. Med. 130, 328. 2. Randerath, K. (1963) J. Chromatogr. 10, 235. 3. Saukkonnen. J. J. (1964);~ Chromatographic Reviews (Lederer. M.. ed.), Vol. 6, p. 53. Elsevier, Amsterdam/New York. 4. Gerlach. E. (1965) J. Chromatogr. 18. 81. 5. Fink, K. (1966) 1. Chromarogr. 22, 118. 6. Silver, M. J., Rodalewicz. J.. Duglas, Y.. and Park, D. (1970) Anal. Biochem. 36, 525. 7. Pataki. G. (1967) J. Chromatogr. 29. 126. 8. Pataki, G. (1967) J. Chromatogr. 29, 133. 9. Lust, J., and Sahud, M. A. (1972) J. Chromatogr. 71, 127. 10. Reyes. Ph. (1972) Anal. Biochem. 50, 35. 11. Jones. C. E. (1972) J. Chromarogr. 64, 378. 12. Hurlbert, R. B.. Schmitz. H., Brumm. A. F., and van Potter, R. (1954)5. Biol. Cl7ern. 209, 13. 13. Cohn. W. E., and BoIIum. F. J. (1961) Biochim. Biophys. Acra 48, 588. 14. Lerner. J. R.. and Shepartz, A. J. I 1968) J. Chromatogr. 35, 37. 15. Davidek, J., Velisek. J., Grundova. K., and Janicek, G. (1971) Z. Lebensnr. Untersc. Forsch. 146, 83. 16. Sweetman. L.. and Nyhan. W. L. (1968) J. Chromatogr. 32, 662. 17. Wasternack. C.. and Reinbothe, H. (1970) J. Chromarogr. 48, 551. 18. Kern, E. D. (1959) Biochim. Biuphys. Acta 32, 554. 19. Nilson, R.. and Sjunnesson. M. (1961) Acta C/rem. Stand. 15, 1017. 20. Davey, C. L. (1962) Biochim. Biophys. Acra 61, 538. 21. Staehlin, M. (1961) Biochim. Biophys. Acta 49. II. 22. Caldwell, J. C. (1969) /. Chromafogr. 44, 331. 23. Rustum, Y. M., and Schwartz, H. S. (1973) Antrl. Biochem. 53,441. 24. Christianson, D. D.. Paulis. J. W., and Wall. J. S. (1968) Anal. Biochem. 22, 35. 25. Lemer, J. R.. Dougherty, Th.M.. and Shepartz. A. J. (1968) J. Chromafogr. 37, 453. 26. Dougherty, Th.M.. and Shepartz, A. J. (1969) J. Chromafogr. 40, 299. 27. Carisano, A.. Riva, M., and Bonchecchi, A. (1969) .I. Chromarogr. 40, 386. 28. Blattner, F. R., and Erickson, H. P. (1972) Anal. Biochem. 18, 220. 29. Brown, Ph.R. (1970) J. Chromatogr. 52, 257. 30. Brown, Ph.R. (1971) 3. Chromatogr. 57. 383. 31. Drobishev. V. J.. Mansurova, S. E., and Kolaev. J. S. (1972) J. Chromatogr. 69, 317. 32. Simon, E. J., and Shemin, D. (1953) J. Amer. Chem. Sot. 75, 2520. 33. Shapiro, H. S. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.). Vol. 12, Part B, p. 205, Academic Press, New York. 34. Williamson, D. H., and Scopes, A. W. (1960) Exp. Cell Res. 20, 238.
BIOCHEMISTRY
81,
136- 142 (1977)
Chromatographic Separation and Identification of Nucleotides from Animal Tissues and Yeast Cells on a Polyethyleneimine Cellulose Column K.-H. lnstittrrr
fiir
Pharntahologir Laltnberge
urrd 355,
PFL~~GER
Tu.rikologie MarbtrrglLabn.
der
Pltilipps-Uttir,ersitrit West
Marburg.
Germany
Received July 13, 1976: accepted March 28. 1977 A procedure has been developed for the separation and identification of nucleotides by anion-exchange chromatography. The method includes column chromatography on PEI-cellulose with an unbuffered exponential salt gradient. This easily handled procedure gives a high degree of resolution and reproducibility and demands no personal attendance during elution. Application of the method is demonstrated with a mouse liver extract and extracts of yeast cells (Saccltarowtyces
cere~‘i.viae).
For separation of nucleotide mixtures obtained from different tissues by acid extraction, a great number of procedures have been developed employing chromatographic techniques on paper (l-6), on thin-layer plates (7-ll), and on columns (12-31), using numerous materials like Dowex (12-15), Sephadex (16,17), ECTEOLA-cellulose (18,19), TEAEcellulose (20), DEAE-cellulose (21-231, PEI-cellulose (23,24), PVP (25, 26), and cation exchangers (27,28). Some of these procedures are very suitable for special problems, such as the purification of nucleotides (25, 26) or the separation of mixtures containing a small number of compounds (15- 17.25-28). Unfortunately, all these techniques revealed several disadvantages when complex mixtures of soluble tissue nucleotides were used and when as many possible individual constituents of the mixture were to be resolved in one run. Chromatographic techniques on thin-layer plates are rapid and relatively easy to carry out. Even with complicated mixtures, they give good separation (7- 11). They are, however, unsuitable for a comprehensive quantitative analysis of unlabeled tissue extracts because of the high salt concentration and the variation in the composition of these extracts. Because of its greater sample capacity, column chromatography is far better suited for such problems, in particular for those conditions in which the largest possible number of individual constituents is to be measured. The techniques of column chromatography described so far are carried out 136 ISSN 0003.2697
NUCLEOTIDE
SEPARATION
ON
A PEI COLUMN
137
with 1300-6000 ml of acidic elution buffer and 30-60 hr of separation time (I?- 15, 22-24). Under these conditions, certain acid-labile materials can be destroyed. Furthermore, as many as five different concentration gradients are used for the separation process (12- 15.22). thereby requiring the presence of supervisory personnel. Cumbersome and costly high-pressure liquid column chromatography requires only 1-6 hr (29-31) for a separation, but uses polystyrene ion exchangers. This does not give good separation when tissue extracts are analyzed, as phenolic substances cause nonionic interactions with these resins. In view of these problems. a column chromatographic method was developed which makes possible a quantitative and reproducible resolution of acid-soluble unpurified tissue extracts, using only one neutral unbuffered NaCl or LiCl concentration gradient. PEI-cellulose was selected as an anion exchanger, as this material gives by far the best separation for nucleotide mixtures on thin-layer plates (9- 11). The method described here uses concave gradients, achieves a high sensitivity to detection using an elution volume of only 300 ml, and separates tissue extracts into as many as 25 symmetrical peaks in 16-20 hr. This method thus makes possible a comprehensive quantitative analysis of individual constituents, without the presence of supervisory personnel during the separation process. Furthermore, this method is less susceptible to complications and requires a very simple preparation of the column. METHODS C’hemicrrls. Nucleotides, nucleosides, nucleobases, UDP-glucose. ADP- and UDP-glucuronate, ApA, NAD, NADP, riboflavin 5phosphate, coenzyme A, folic acid, and riboflavin were purchased from Boehringer, Mannheim, W. Germany. [6-3H]uracil and [Ylinosin were obtained from Amersham Buchler, Braunschweig, W. Germany. OMP was from CalBiochem, Luzern, Switzerland. Acetylcoenzyme A and the mixture of oligonucleotides were prepared in our laboratory by methods described by Simon and Shemin (32) and Shapiro (33), respectively. PEI-Cellulose and the other chemicals were obtained from E. Merck, Darmstadt, W. Germany. All solutions were prepared with deionized glass-distilled water. Prepur-ation of the ion e.uchangc~r. PEI-Cellulose, 50.0 g, was vigorously stirred in 200 ml of 1 M NaCl. Then, the cellulose was allowed to sediment. and the yellowish overlay was siphoned off. In this manner, the washing was repeated four or five times. The sediment was then suspended in about 100 ml of 1 M NaCl and was carefully removed. This slurry is suitable for filling the columns, but can also be stored, well sealed, in the dark for up to 4 weeks without any loss of quality.
138
K.-H.
PFLLIGER
The column is made of cylindrical glass tubing (0.9 x 25 cm) which is tapered off at both ends. The outlet is connected with a thin silicone tube. It has a capacity of about 7.0 g of PEI-cellulose (dry weight), adsorbing a total of 1.5 mol of ATP. The top of the column is connected to a funnel which allows a hydrostatic pressure of 40 cm. The valve is closed, and the tapering outlet is plugged with a wet pea-sized piece of glass wool. The column is filled with evacuated I M NaCl, and the prepared ion exchanger is poured into the funnel. After several hours of settling, the valve is opened, and the column is allowed to drip with a hydrostatic pressure of 40 cm for 4 hr. Now, the excess ion exchanger is carefully Preparation
of the column.
Tronsmlttance 4 30
Molortty
at 260 nm
of NaCl
PH 8.0
LO.
6.0
50
L.0.
1
30 2.0. 1.0. L
gradlent
6
6
IO
12
11
16
16
20
22 Tune lhl
elution
Absorbance 1‘ 1.2 1.0
i
&gin of gradlent elurlo” , AMP
,ADP
ATP
0.6. 0.6.
FIG. 1. (a) Chromatography of an extract of 250 mg (wet weight) of yeast cells (Saccharomyces cerevisiue) previously grown in Williamson and Scopes (34) medium. The yeast was harvested on a membrane filter, washed with ice-cold water. and extracted for IS was neutralized with potassium min with ice-cold 0.6 M perchloric acid at 2°C. The extract hydroxide and was centrifuged to remove the cell residue and the potassium perchlorate. The neutral extract was used immediately for chromatography or was lyophilized for storage. For the numbers of the peaks, see Table 1. (b) Chromatography ofa mouse liver extract. The mice were killed. and the livers were rapidly excised with pliers precooled in liquid nitrogen. The liver was completely frozen in nitrogen and was shattered in a steel mortar. The extraction and the following preparation were similar to the procedure described for yeast cells, except that the cell residue was removed before neutralization.
NUCLEOTIDE
SEPARATION
ON
A PEI COLUMN
139
siphoned off, the funnel is cleaned, and the column is equilibrated with glass-distilled water for at least 5 hr. The column is then ready for use. It is possible to regenerate the columns after operation by washing them for 4 hr with water. Thus it is possible to use them for as many as five separations. Unfortunately, the resistance to Bow increases with each operation of the column. Usually, the columns were used for only one analysis. Starzdard elution system. At the beginning, the mixing chamber contains 240 ml of glass-distilled water. NaCl, 4.15 M was pumped into the mixing chamber at a flow rate of 4.0 mlihr, while the elution flow was maintained at 16.0 ml/hr by a metering pump (Fa. Biihler. Tiibingen, W. Germany). The duration of gradient elution was 18-20 hrs: the elution volume was about 300 ml. Ehtion arzd identi$cation of eluted materials. For initial chromatographic studies, nucleotide mixtures composed of lo- 16 compounds (100 pg of each nucleotide) are applied to the columns in 2-3 ml of water. The neutralized extracts from 150-2.50 mg (wet weight) of animal tissues or yeast cells are dissolved in 5- 10 ml of water. After absorption of the solutions, the column is washed for 2-3 hr with water at a flow rate of 16 ml/hr to remove ribonucleosides, bases, and other nonanionic compounds. Now. the columns are eluted with the exponential salt gradient. The eluates are monitored at 257 or 260 and 280 nm synchronously, and the collected nucleotide materials in each elution peak were identified on the basis of the following criteria. (i) Ultraviolet absorption spectra of the pooled peaks at ph 2.0, 7.0, and 12.0 were determined on a Zeiss DMR 21 spectrophotometer: (ii) coincident elution from the column with authentic samples added to the tissue extracts: (iii) The elution patterns of radioactively labeled extracts by incorporation of [6-“Hluracil or [‘“Clinosine into living cells. RESULTS
Under the described condition. following equation:
AND DISCUSSION
the exponential L.,n , -“A A -1
salt gradient yields the
L’ IIs, I.
where t (min) = time; A (ml. min-‘) = operation flow rate; Z (ml. min-I) = flow rate of salt solution pumped into the mixing chamber: M, (ml) = volume of water in the mixing chamber at the point of time t = 0: K, (mol. 1-l) = concentration of the added salt solution; K, (mol. l-‘) = salt concentration of the gradient at point of time t.
140
K.-H. Absorbance
PFLOGER
ot 260 “In
@agin of gradlent
elutnn
FIG. 2. Chromatography of an extract ously exposed to [3H]uridine (0.1 PCiiml) were extracted by the usual procedure.
Volume of
eluate
Cm!1
of yeast cells (Succhcrvonzyceu rere~Giae) previin Williamson and Scopes (34) medium. The cells
In this paper are specified the data of the standard gradient which was found most acceptable for the resolution of tissue extracts. The length, the slope, and the final height of the gradient can be varied for special separation problems by varying M,,, and/or K,, and/orZ, and/or A.
The results of chromatographic separation of extracts from yeast cells and liver tissue, using the standard gradient, are shown in Figs. la, b and 2. Table 1 presents the retention times and the localization of more than 40 standard substances by coincident elution from the column with yeast-cell extracts. The high degree of resolution and reproducibility has been already documented by more than 400 chromatograms of complex mixtures such as tissue extracts. Rustum and Schwartz (23) have described a useful separation method for acid-soluble tissue extracts on DEAE-cellulose. In a comparison of this matrix with PEI-cellulose, they found that the latter could not be stored as easily and showed a considerable batch variability, thus giving only incomplete separations. These disadvantages of PEI-cellulose were overcome by using the simple method for preparation of the commercial material and a standard column preparation method, with the result that PEI-cellulose, in all respects, proved to be significantly superior to DEAE-cellulose in work with neutral eluents. The standard nucleotides like mono-, di-, and triphosphates were recovered in a yield of 92-101%. Only ADP gave a yield of 78%. The fractions are eluted in volumes of 5-15 ml involving a high sensitivity to detection and a relatively small contamination by salt. When NaCl is replaced by LiCl, the salt can be removed by ethanol extraction. When the
NUCLEOTIDE
SEPARATION TABLE
RLTENTION
Retention thr)
with
Gradient 1.97 1.13 2.43 2.9 3.3 3.6 3.83 4.43 4.76
elution
5.83 6.3 6.5 6.43 7.43 7.63 8.8 9.9 10.7 II.0 II.5 17.13 17.9 13.7 14.2 14.6 15.2 16.8
141
A PEI COLUMN
I
TIMES DFTHE CohlPouNos OF YEAST AND STANDARD SURSl'ANCTS
time
Washing O-2.0
ON
Localization of standard substances
EXTRACTS
No. of elution peak
H,O Bases. nucleosides, acids. riboflavin. pyridoxine
Orotic NAD, CAMP
acid ApA,
amino aneurine.
oligonucleotides
I la ?
UDP-Glucose ADP-Glucose UMP. dTMP CMP. dCMP 5’-AMP. 3’-AMP. 2’.AMP. dAMP GMP, dGMP (II Maximum of riboflavin-PO, peak (2) Maximum of riboflavin-PO, peak UDP-Glucuronate NADP OMP. folic acid UDP CDP ADP GDP Acetyl-CoA UTP, dTTP CTP. ITP ATP CoA-SH GTP Adenosine tetraphosphate, RNA fractions
3 4 4a 5 6 I 8 8a Xa 9 IO II I:! I3 I4 I5 I6 l6a I7 l7a I8 I9
pH of the eluate is maintained in the range of pH 6.5-7.0. acid-labile materials are hardly destroyed during chromatography. Only during the elution of acidic material like amino acids, folic acid, etc., do values as low as pH 4.0 occur. This simple and practical procedure offers a number
142
K.-H.
PFLiiGER
of advantages over existing methods and should permit further studies of the nucleotide metabolism. ACKNOWLEDGMENTS For calculation of the equation. the author would like to thank Mr. F. J. Thome, Institut fur medizinische und biologische Statistik Marburg, W. Germany.
Mr. G. Albricht and und Dokumentation.
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