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Ion-exchange chromatography of nucleotides on polyethyleneimine cellulose columns: Analysis of maize grain extracts
ANALYTICAL
BIOCHEMISTRY
22,
Ion-Exchange on
3546
Chromatography
Polyethyleneimine Analysis
Regional
of Cellulose
of Maize
D. D. CHRISTIANSON, Northern
(1968)
Grain
Received
Columns: Extracts1
J. W. PAULIS,
Research Laboralory,’ March
Nucleotides
Peoria,
AND
J. S. WALL Illinois
61604
8, 1967
Free nucleotides are difficult to isolate and identify in complex mixtures such as in extracts from plant tissues. The ion-exchange chromatographic techniques initiated by Cohn (1) made possible the separation of a large nu’mber of nucleotides and their derivatives. The nucleotides from plant materials have been chromatographed on polystyrene anionexchange resins with various eluants (2-4). However, large amounts of ultraviolet-absorbing phenolic contaminants in the extract interfered with the chromatographic analysis on the ion-exchange resins. Preliminary purification of such extracts was necessary but resulted in loss of nucleotides. In the course of separating nucleotides isolated from maize at various degrees of maturity, similar difficulties were encountered when polystyrene anion-exchange resins were used. Therefore, other ionexchange materials for separating nucleotides were investigated. Alternatives to the polystyrene resins, to which phenolic substances adhere, are anion-exchange celluloses. These materials hare been used to purify oligonucleotides from nucleic acid digests and some resolution of mononucleotides was obtained (5, 6). Separations of mono-, di-, and triphosphate nucleotides on ECTEOLA (7)) TEAE (8)) and DEAE (9, 10) celluloses have also been investigated, but the resolution of complex nucleotide mixtures with the commercial adsorbents then available was not satisfactory. Randerath (11) introduced polyethyleneimine (PEI) cellulose for thin-layer chromatographic separation of nucleotides. Christianson et al. (12) demonstrated that resolution and reproducibility of nucleotide separations were improved when a microcrystalline cellulose ‘Presented at the Division of Biological Chemistry, 152nd Meeting, Chemical Society, New York, New York, September 1966. *A laboratory of the Northern Utilization Research and Development Agricultural Research Service, U. S. Department of Agriculture. 35
American Division,
36
CHRISTIANSON,
PAULIS,
AND
WALL
(Avicel)3 was substituted for the standard fibrous Cellulose previously used with PEI in thin-layer chromatography. In the work presented here PEI-Avicel was employed as the ion exchanger in column chromatographic separations. The nucleotide mixtures were resolved with a gradient elution system permitting independent pH and ionic strength changes and borate complexing of the sugar substituents. The procedure was applied to analysis of complex mixtures of nucleotides in extracts of maize. METHODS
Preparation of the Ion Exchanger
An aqueous solution of 50% PEP was neutralized to pH 7, dialyzed against 3 liters of water for 20 hr, and diluted to a 1% solution in a manner similar to the procedure of Randerath (13). In a Waring Blendor, 25 gm of Avicel (38 p bead size) was mixed for 1 min at full speed with 150 ml of the 1% PEI solution. The partially gelled mix was then layered to a thickness of 0.25 in. in glass trays and dried at room temperature for 62-96 hr in the dark. The dried material (moisture content 5-7%) was ground in a Wiley mill, intermediate model, using a 60-mesh screen. The fine powder was washed with absolute methanol to remove excess PEI and dried in air. The PEI-Avicel, containing 1.0 f 0.05 meq nitrogen/gm, served as column packing for the chromatographic separations. Nitrogen content was determined by semimicro-Kjeldahl analysis. By mixing solutions of PEI of different concentrations with Avicel in the same volume-weight ratio, exchange capacities were altered. Development of a Gradient Elution System for the Separation of Nucleotides
Nucleotides were chromatographed on 0.9 X 100 cm columns of the anion exchanger (Avicel prepared with 1% PEI) . The columns were packed in sections with 50 ml slurries of a 1:l ratio (w/vj of the exchanger to water. Flow rates of 50 ml/hr were #maintained with a MiltonRoy minipump during packing of the columns. Columns were rinsed with several bed-volumes of water before chromatography. Fresh columns were poured for each analysis. Reuse of columns was not investigated. In the initial chromatographic studies, nucleotide mixtures (0.5 to 3 PEI is available from Chemirad Corporation, P.O. Box 187, East Brunswick, New Jersey, or Badische Anilinund Sodafabrik, Ludwigshafen am Rhem, Germany. Avicel is available from The FMC Corporation, American Viscose Division, Marcus Hook, Pennsylvania. Mention of suppliers of chemicals or equipment does not constitute preferential endorsement of their products by U. S. Department of Agriculture.
CHROMATOGRAPHY
OF
37
NUCLEOTIDES
1.0 @mole) of 3’-UMP, 5’-CMP, 5’-AMP, 5’-GMP, ADP-glucose and UDP-glucose were applied to the columns in 2-3 ml water. The nucleotides were eluted with either 0.6M LiCl at pH 7.0 or 0.6M LiCl at pH 4.5 to determine influence of pH, LiCl concentrations, and borate concentrations on their elution. The columns were operated at a flow rate of 50-80 ml/hr
and Chromatography
of Immature
Maize Grain Extracts
Maize plants were hand-pollinated in the field and cobs picked 25 days after pollination. Kernels were shelled from the cobs after quick freezing in a solid carbon dioxide-ethanol bath and immediately lyophilized to dryness. The dried material was finely ground in a RobinsMyers bench top hammer mill through a 0.027-m. mesh screen. A 25 gm sample was extracted by stirring with 100 ml 10% trichloroacetic acid (TCA) at 4°C and then centrifuged at 4°C. The residue was rcextracted with 100 ml 5% TCA and centrifuged. The final residue was washed with 50 ml 5% TCA. Immediately after each treatment the TCA in the extracts and wash was removed by extraction with ether at 4°C until the solutions were almost neutral. All aqueous extracts were combined and lyophilized. A portion of the dried lyophilized extract containing ultraviolet-absorbing substances equivalent to 750 absorbance units at 260 rn,p was dissolved in 2-5 ml water and applied to a 1% PEI-Avicel column without further purification. The column was washed with water until the ultraviolet absorbance of t.he effluent nas less t.han 0.1. The extract was then chromatographed with the complex gradient elution system developed for the standard nucleoside mono- and diphospbate mixture. Characterization
of Nucleotides
Fractions comprising the central region of a peak were pooled, neutralized to pH 7, and lyophilized. Eluant buffer salts were removed by
38
CHRISTIANSON,
PAULIS,
AND
WALL
extraction with an acetone-ethanol mixture (4:l) as described by Blumsom and Baddiley (15). Standard nucleotides were recovered in yields of 90-95s with little degradation of sugar nucleotides. After desalting, the pooled fractions were checked for purity by paper (16) or by thinlayer chromatography (17). In most instances, the fractions consisted of a single nucleotide, which could be characterized directly without further purification. Where peaks overlapped, nucleotides were separated by preparative paper chromatography with solvent systems of Paladini and Leloir (16). Ultraviolet absorption spectra of the nucleotides at neutral pH were determined on a Cary recording spectrophotometer and concentrations calculated from their extinction coefficients. Reducing values of the sugars liberated from the sugar nucleotides after hydrolysis with 0.01 N HCl for 10 min at 100°C were determined by the method of Park and Johnson (18). Total carbohydrate was estimated by the phenol-HSO, method (19). Ribose content of the nucleotides was determined by the orcinol method (20). 5-Adenylic and 5uridylic acid served as standards for purine and pyrimidine nucleotides, respectively. Total phosphorus content was estimated by the method of Hurst (21). The base moieties of the nucleotides were liberated by hydrolysis in 3N HCl for purine nucleotides (22) or in concentrated formic acid for pyrimidine nucleotides (23). The bases were isolated by paper chromatography and characterized by spectral analysis. To confirm base characterization, total nitrogen content was determined by Nesslerization as modified by Middleton (24). RESULTS AND DISCUSSION Properties
of PEI-Avid
The amount of PEI associated with Avicel as determined by nitrogen content depended upon the concentration of PEI in the initial mixture as illustrated in Figure 1. The adherence of PEI to Avicel was also significantly influenced by the length of drying time. To retain maximum sorption of PEI, it was necessary to dry the material below 7% moisture before washing with methanol. Removal of excess PEI was more efficient with absolute methanol than with water because of greater PEI solubility in the alcohol. Washing with methanol also contributes to fixation of polymer to the microcrystalline bead. When water was used for washing, leaching of PEI occurred during chromatography. Mixes containing more than 37% PEI were unstable when prepared as above. As indicated in Figure 2, acid-binding capacity was used as a criterion for measuring ion-exchange sites of the prepared batches of 1 and 3% PEI-Avicel (curves C and D, respectively). To obtain the curves, PEI-
CHROMATOGRAPHY
39
OF NUCLEOTIDES
Avicel (40 mg) in 5 ml of H,O was titrated to pH 12.0 with 0.5 1M NaOH and titrated back to pH 3.0 with 0.12 M HCl. Avicel was run also as a control to establish the amount of excess OH- added at pH 12 and also to compensate for the a’mount of acid that will bind with Avicel alone (curve L4). The milliequivalents of acid was then subtracted from curve A to determine the number of ion-exchange sites available. For example, the ion-exchange capacity of the preparation illustrated in curve C was 0.8 meq/gm in excess of that of the Avicel alone. The 3.0 5 2.5\” 5 2.0 z” f g 1.5d I 1.0 -
0/ 0/ p/
Z E 0.5 II
FIG. 1. Sorption Mixture was dried determination.
I 1.0
I 1.5
I 2.0 PEI, %
of PEI by Avicel concentrations for 96 hr and washed extensively
I 2.5
I 3.0
of PEI mixed with Avicel. with methanol before nitrogen
nitrogen content of the same preparation was 1.0 mmole/gm (Fig. 1). Approximately 80% of the PEI nitrogen that adhered to Avicel was available for ion exchange. The ion-exchange capacity of this PEIAvicel was considerably higher than that of the sample of commercial PEI-cellulose tested (curve B) . Selection of Gradient El&ion of Standard
System and Chromatography Nucleo tides
Figure 3 illustrates the influence of pH in maintaining peak height during chromatography and the influence of borate ion complexing on the elution position of nucleotides. The common 5’-monophosphates and
40
CHRISTIANSON,
PAULIS,
AND
WALL
3’-monophosphate of uridine plus UDP-glucose and ADP-glucose were eluted from 0.9 X 100 cm columns of Avicel prepared with 1% solution of PET under conditions described. In chromatogram A at pH 7 the sharpness of the peaks was diminished as the elution progressed. Consequently, it was necessary to increase protonixation of the ion exchanger by eluting with 0.6M LiCl at pH 4.5 (chromatogram B). The elution position of nucleotides was retarded since the column was not pre-
I 0
I 1
I I 2 3 Acid, meq.
I 4
I
5
FIQ. 2. Titration curves of PEI-Avicel showing pH change vs. meq acid added per gram: (A) Avicel only, (B) commercial PEI-cellulose, (C) PEI-Avicel made with 1% PEI (used for chromatography), (D) PEI-Avicel made with 3% PEI.
equilibrated ; however, the peak heights were ‘maintained under these conditions. The advantage of protonization was incorporated in the system used in chromatogram C. A pH gradient obtained with boric acid maintains sharpness of the nucleotides eluted last. Note the change in position of the sugar nucleotides. The pronounced effect of borate ions on the anion-exchange behavior of sugar nucleotides has been described (25).
CHROMATOGRAPHY
OF
41
NUCLEOTIDES
The final gradient elution system selected for routine use is shown in Figure 4. To ensure borate complexing of sugar nucleotides, deoxyribonucleoside monophosphates, and ribonucleoside monophosphates; pH 6.5 to 7.0 was maintained throughout this area of elution. Wucleoside monophosphates were eluted as sharp peaks with this slight pH change. The pH was then decreased by further addition of boric acid to maintain sharp peaks in the elution area of the nucleoside diphosphates. With this elution system, good resolution of standard 5’-mono- and 5’-diphosphates of uridine, cytidine, adenosine, and guanosine was achieved (Fig. 5). The 3’-UMP was also included in the mixture to demonstrate separation
B. LiCI-HCI pH 4.5
UDPG
ADPG 3’4MP ’
CMP GMP
FIG. 3. Separation of mixture of nucleoside monophosphates to illustrate influence of pH on peak height and borate complexing on relative elution position. All nucleotides are the 5’-isomers with the exception of the monophosphate of uridine, which is the 3’-isomer. Chromatogram B was not preconditioned to pH 4.5 before chromatography. Column: PEI-Avicel made with 1% PEI, 0.9 x 100 cm, bead size 38 m$. Elution system as described: flow rate, 45 ml/hr; O.bl.0 pmole of each substance was applied on column.
of this analog. The sugar nucleotides of glucose, here exemplified by ADP-glucose and UDP-glucose, were also separated. Along with these sugar nucleotides, deoxyribonucleotides were resolved from their ribonucleotide analogs by the presence of borate in the system. This effect was described previously (26), Most of the nucleotides were well resolved in a small volume (20-50 ml). This system was most satisfactory for a survey of the nucleotides in immature maize. For resolution of poorly resolved areas, rechromatography of desalted peaks with different gradient systems has been successful.
42
CHRISTIANSON,
PAULIS,
AND
WALL
Evidence has been obtained by thin-layer chromatography that PEIAvicel having higher exchange capacities than those employed here gives better resolution of nucleoside monophosphates. Higher capacity PEI-Avicel may be advantageous for use in column chromatography when these compounds are of major interest. Nucleoside triphosphates can also be separated on these columns by extending the elution gradient. Degradation occurs, however, during the time necessary for their elution. Separate chromatography on shorter
7.0
1.6 tt
Volume, ml.
FIG. 4. Gradient elution system of LiCl and HBOa for separation of mono- and diphosphate nucleotides based on six chambers (350 ml each) of a nine-chambered Varigrad. Chambers 1 and 2 contained H,O; chambers 3, 4, 5, and 6 contained 0.50, 2.0, 2.0, 2.0 A4 concentrations of LiCl and 0.526, 0.132, 0.395, and 0.526 M concentrations of HJSO,, respectively. The pH of the system during chromatography is illustrated.
columns, where more rapid elution results, diminished this degradation. Purine and pyrimidine bases and nucleosides do not interfere with chromatography of nucleoside monophosphates on PEI-Avicel. Their secondary binding interactions with this cellulose appear to be minimal. They are eluted very early, in contrast to their behavior on polystyrene resins, where they elute in some cases in the vicinity of some nucleoside monophosphates.
CHROMATOGRAPHY
Chromatography
of Immature
OF
KUCLEOTIDES
43
Maize Grain Extracts
Figure 6 shows the application of this chromatographic procedure for the separation of free nucleotides isolated from immature maize grain (25 days after pollination). The acid-soluble nucleotides were chromatographed directly without preliminary purification of the extract. Direct analysis of the extracts reduces degradation or losses of the nucleotides, especially sugar nucleotides. Use of charcoal adsorption and desorption with alkaline systems, or more extensive liquid-liquid ether extractions
FIQ. 5. Ion-exchange separation of a mixture of 5’-mono- and diphosphates of adenosine, guanosine, uridine, and eytosine, and also including 3’-monophosphate of uridine ; and adenosine diphosphate (ADP)-glucose (Glu), uridine diphosphate (UDP)-glucose (Glu). Guanine and guanosine were also added to the mixture to illustrate their elution positions. Column : PEI-Avicel made with 1% PEI, 0.9 x 100 cm, bead size 38 p. Elution system: simultaneous gradients of LiCl and HB08 with variable pH as shown in Figure 4. Column was washed with 175 ml Hz0 before start of gradient. Only LiCl gradient illustrated in this figure. Flow rate 45 ml/hr; 0.5-1.0 FmoIe of each substance was applied on column.
or both, for purification of the extract is avoided. The ultravioletabsorbing contaminants which these techniques seek to eliminate interfere with the chromatography on polystyrene resins. We observed that nucleotides in extracts of maize grain were not well resolved from residual phenolic materials on polystyrene resins even after preli,minary partial purification. Most of these nonnucleotide ultraviolet-absorbing materials were not adsorbed to the PEI-Avicel. Separation of these contaminants from nucleotides was achieved on PEI-Avicel columns by their elimination in the initial water wash. As much as 70% of the total absorbance applied to the column can be eliminated by washing. The
44
CHRISTIANSON,
PAULIS,
AND
WALL
low-baseline ultraviolet absorbance achieved throughout the run demonstrates further that little ultraviolet absorbing material is smeared through the column during fractionation. Even in the extreme case of mature maize extracts which contain higher contents of nonnucleotide materials including phenolic substances, an extended period of pre1.00 -’
- 0.75 2 z 3 0.50 % Z 2e 0.20 -
0
100
500
1000
Effluent, ml.
FIG. 6. Separation of acid-soluble nucleotides of immature maize grain (25 days after pollination). Chromatographic conditions similar to those described in Figure 5. Peaks, 5, 7, 8, and 10 were identified as UPD-X, GDP-X, 5’UMP, and 5’AMP, respectively. See Table 1 for details of characterization.
li,minary washing of the column also resulted in low blank values with well resolved peaks. Four of the major peaks from this separation were desalted and their composition was investigated. Analysis of the substituents of these nucleotides were performed directly on the desalted fraction -without further purification. Table 1 gives the analytical data compiled for their TABLE 1 Chemical Analysis of Nucleotides Isolated from Immature Composition, ‘E?
filll&S
5 7 8 10
5.5 11.8 13.3 6.0
““:“,‘e”
Uridine Guanosine Uridine Adenosine
= 25 days after pollination. b Peaks from Figure 6.
Compound identified
UDP-X GDP-X 5’-UMP 5’-AMP
Maize Graina
moles/mole
Total phosphorus
Total nitrogen
Pentose
1.75 2.40 1.20 1.00
1.90 3.72 2.10 5.42
1.08 0.90 0.99 0.86
base Reducing sugar after hydrolysis
1.00 0.96
CHROMATOGRAPHY
OF NUCLEOTIDES
45
identification. The sugar substituents of the two sugar nucleotides are still under investigation. A more detailed study of the variety and changes of nucleotides in separate embryo and endosperm fractions during maturation is in progress. The nucleotide separations possible with this method should permit further study of the role of nucleotides in the biosynthesis of various carbohydrates in the developing seed. SUMMARY
A procedure has been developed for the quantitative chromatographic separation of free nucleotides isolated from maize grain extracts on columns of polyethyleneimine-microcrystalline cellulose. Use of a uniform microcrystalline cellulose provides sharper separations of nucleotides as compared with separations with standard fibrous celluloses. Mono- and diphosphate nucleotides and sugar nucleotides are eluted with a concave gradient elution system of LiCl and boric acid, in which ionic strength and pH are independently changed. Borate complexing facilitates both separation of sugar nucleotides from their related diphosphate nucleotides and separation of deoxyribo- and ribonucleotides. Phenolic materials and other plant constituents that interfere with the detection of nucleotides during chromatography are less readily adsorbed on PEI-cellulose. Use of PEI-cellulose, therefore, permits direct analysis of some plant extracts without extensive preliminary purification, which may result in degradation and loss of nucleotides. Application of the method is illustrated by the analysis of free nucleotides contained in immature ‘maize seeds. REFERENCES 1. COHN, 2.
3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
W. E., J. Am. Chem. Sot. 72, 1471 (1950). BERGKVIST, R., Acta Chem. Stand. 10, 1303 (1956). GINSBURG, V., STUMPF, P. K., AND HASSID, W. Z., J. Biol. Chem. 223, 977 (1956). BROWN, E. G., Biochem J. 85,633 (1962). STAEHELIN, M., PETERSON, E. A., .~ND SOBER, H. A., Arch. Biochem. Biophys. 85, 289 (1959). TOMLINSON, R. V., AND TENER, G. M., Biochemistry 2, 697 (1963). NILSSON, R., AND SJUNNESSON, M., Acta Chem. Stand. 15, 1017 (1961). DAVEY, C. L., Biochim. Biophys. Acta 61, 538 (1962). OCKERMAN, P. A., Biochim. Biophys. Acta 74,588 (1963). STAEHELIN, M., B&him. Biophys. Actu 49, 11 (1961). GNDERATH, K., Angew. Chem. 74, 780 (1962); Intern. Ed. 1, 553 (1962). CHRISTIANSON, D. D., SINCLAIR, H. B., AND PAULIS, J. W., Biochim. Biophys. Actu 121, 412 (1966). RANDERATH, K., Biochim. Biophys. Acta 81,852 (1962). PETERSON, E. A., AND SOBER, H. A., Anal. Chem. 31,857 (1959). BLUMSOM, N. L., AND B.4DDILEY, J., Biochem. J. 81, 114 (1961).
46 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
CHRISTIANSON,
PAULIS,
AND
WALL
PALADINI, A. C., AND LELOIR, L. F., Biochem. J. 51,426 (1952). RANDERATH, K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). PARK, J. T., AND JOHNSON, M. J., J. Biol. Chem. 181, 149 (1949). DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, Chem. 28, 350 (1956). MEJBAUM, W., 2. Physiol. Chem. 258, 117 (1939). HURST, R. O., Can. J. Biochem. 42,287 (1964). DANKERT, M., PASSERON, S., RECONDO, E., AND LELOIR, L. F., Biochem. Res. Commun. 14, 1358 (1964). VISCHER, E., AND CHARGAFF, E., J. Biol. Chem. 186, 715 (1948). MIDDLETON, R. R., J. Appl. Chem. 10,281 (1960). RANDERATH, K., AND RANDERATH, E., Anal. Biochem. 13,575 (1965). KHYM, J. X., AND COHN, W. E., Biochim. Biophys. Acta 15, 139 (1954).
F., Anal.
Biophys.
BIOCHEMISTRY
22,
Ion-Exchange on
3546
Chromatography
Polyethyleneimine Analysis
Regional
of Cellulose
of Maize
D. D. CHRISTIANSON, Northern
(1968)
Grain
Received
Columns: Extracts1
J. W. PAULIS,
Research Laboralory,’ March
Nucleotides
Peoria,
AND
J. S. WALL Illinois
61604
8, 1967
Free nucleotides are difficult to isolate and identify in complex mixtures such as in extracts from plant tissues. The ion-exchange chromatographic techniques initiated by Cohn (1) made possible the separation of a large nu’mber of nucleotides and their derivatives. The nucleotides from plant materials have been chromatographed on polystyrene anionexchange resins with various eluants (2-4). However, large amounts of ultraviolet-absorbing phenolic contaminants in the extract interfered with the chromatographic analysis on the ion-exchange resins. Preliminary purification of such extracts was necessary but resulted in loss of nucleotides. In the course of separating nucleotides isolated from maize at various degrees of maturity, similar difficulties were encountered when polystyrene anion-exchange resins were used. Therefore, other ionexchange materials for separating nucleotides were investigated. Alternatives to the polystyrene resins, to which phenolic substances adhere, are anion-exchange celluloses. These materials hare been used to purify oligonucleotides from nucleic acid digests and some resolution of mononucleotides was obtained (5, 6). Separations of mono-, di-, and triphosphate nucleotides on ECTEOLA (7)) TEAE (8)) and DEAE (9, 10) celluloses have also been investigated, but the resolution of complex nucleotide mixtures with the commercial adsorbents then available was not satisfactory. Randerath (11) introduced polyethyleneimine (PEI) cellulose for thin-layer chromatographic separation of nucleotides. Christianson et al. (12) demonstrated that resolution and reproducibility of nucleotide separations were improved when a microcrystalline cellulose ‘Presented at the Division of Biological Chemistry, 152nd Meeting, Chemical Society, New York, New York, September 1966. *A laboratory of the Northern Utilization Research and Development Agricultural Research Service, U. S. Department of Agriculture. 35
American Division,
36
CHRISTIANSON,
PAULIS,
AND
WALL
(Avicel)3 was substituted for the standard fibrous Cellulose previously used with PEI in thin-layer chromatography. In the work presented here PEI-Avicel was employed as the ion exchanger in column chromatographic separations. The nucleotide mixtures were resolved with a gradient elution system permitting independent pH and ionic strength changes and borate complexing of the sugar substituents. The procedure was applied to analysis of complex mixtures of nucleotides in extracts of maize. METHODS
Preparation of the Ion Exchanger
An aqueous solution of 50% PEP was neutralized to pH 7, dialyzed against 3 liters of water for 20 hr, and diluted to a 1% solution in a manner similar to the procedure of Randerath (13). In a Waring Blendor, 25 gm of Avicel (38 p bead size) was mixed for 1 min at full speed with 150 ml of the 1% PEI solution. The partially gelled mix was then layered to a thickness of 0.25 in. in glass trays and dried at room temperature for 62-96 hr in the dark. The dried material (moisture content 5-7%) was ground in a Wiley mill, intermediate model, using a 60-mesh screen. The fine powder was washed with absolute methanol to remove excess PEI and dried in air. The PEI-Avicel, containing 1.0 f 0.05 meq nitrogen/gm, served as column packing for the chromatographic separations. Nitrogen content was determined by semimicro-Kjeldahl analysis. By mixing solutions of PEI of different concentrations with Avicel in the same volume-weight ratio, exchange capacities were altered. Development of a Gradient Elution System for the Separation of Nucleotides
Nucleotides were chromatographed on 0.9 X 100 cm columns of the anion exchanger (Avicel prepared with 1% PEI) . The columns were packed in sections with 50 ml slurries of a 1:l ratio (w/vj of the exchanger to water. Flow rates of 50 ml/hr were #maintained with a MiltonRoy minipump during packing of the columns. Columns were rinsed with several bed-volumes of water before chromatography. Fresh columns were poured for each analysis. Reuse of columns was not investigated. In the initial chromatographic studies, nucleotide mixtures (0.5 to 3 PEI is available from Chemirad Corporation, P.O. Box 187, East Brunswick, New Jersey, or Badische Anilinund Sodafabrik, Ludwigshafen am Rhem, Germany. Avicel is available from The FMC Corporation, American Viscose Division, Marcus Hook, Pennsylvania. Mention of suppliers of chemicals or equipment does not constitute preferential endorsement of their products by U. S. Department of Agriculture.
CHROMATOGRAPHY
OF
37
NUCLEOTIDES
1.0 @mole) of 3’-UMP, 5’-CMP, 5’-AMP, 5’-GMP, ADP-glucose and UDP-glucose were applied to the columns in 2-3 ml water. The nucleotides were eluted with either 0.6M LiCl at pH 7.0 or 0.6M LiCl at pH 4.5 to determine influence of pH, LiCl concentrations, and borate concentrations on their elution. The columns were operated at a flow rate of 50-80 ml/hr
and Chromatography
of Immature
Maize Grain Extracts
Maize plants were hand-pollinated in the field and cobs picked 25 days after pollination. Kernels were shelled from the cobs after quick freezing in a solid carbon dioxide-ethanol bath and immediately lyophilized to dryness. The dried material was finely ground in a RobinsMyers bench top hammer mill through a 0.027-m. mesh screen. A 25 gm sample was extracted by stirring with 100 ml 10% trichloroacetic acid (TCA) at 4°C and then centrifuged at 4°C. The residue was rcextracted with 100 ml 5% TCA and centrifuged. The final residue was washed with 50 ml 5% TCA. Immediately after each treatment the TCA in the extracts and wash was removed by extraction with ether at 4°C until the solutions were almost neutral. All aqueous extracts were combined and lyophilized. A portion of the dried lyophilized extract containing ultraviolet-absorbing substances equivalent to 750 absorbance units at 260 rn,p was dissolved in 2-5 ml water and applied to a 1% PEI-Avicel column without further purification. The column was washed with water until the ultraviolet absorbance of t.he effluent nas less t.han 0.1. The extract was then chromatographed with the complex gradient elution system developed for the standard nucleoside mono- and diphospbate mixture. Characterization
of Nucleotides
Fractions comprising the central region of a peak were pooled, neutralized to pH 7, and lyophilized. Eluant buffer salts were removed by
38
CHRISTIANSON,
PAULIS,
AND
WALL
extraction with an acetone-ethanol mixture (4:l) as described by Blumsom and Baddiley (15). Standard nucleotides were recovered in yields of 90-95s with little degradation of sugar nucleotides. After desalting, the pooled fractions were checked for purity by paper (16) or by thinlayer chromatography (17). In most instances, the fractions consisted of a single nucleotide, which could be characterized directly without further purification. Where peaks overlapped, nucleotides were separated by preparative paper chromatography with solvent systems of Paladini and Leloir (16). Ultraviolet absorption spectra of the nucleotides at neutral pH were determined on a Cary recording spectrophotometer and concentrations calculated from their extinction coefficients. Reducing values of the sugars liberated from the sugar nucleotides after hydrolysis with 0.01 N HCl for 10 min at 100°C were determined by the method of Park and Johnson (18). Total carbohydrate was estimated by the phenol-HSO, method (19). Ribose content of the nucleotides was determined by the orcinol method (20). 5-Adenylic and 5uridylic acid served as standards for purine and pyrimidine nucleotides, respectively. Total phosphorus content was estimated by the method of Hurst (21). The base moieties of the nucleotides were liberated by hydrolysis in 3N HCl for purine nucleotides (22) or in concentrated formic acid for pyrimidine nucleotides (23). The bases were isolated by paper chromatography and characterized by spectral analysis. To confirm base characterization, total nitrogen content was determined by Nesslerization as modified by Middleton (24). RESULTS AND DISCUSSION Properties
of PEI-Avid
The amount of PEI associated with Avicel as determined by nitrogen content depended upon the concentration of PEI in the initial mixture as illustrated in Figure 1. The adherence of PEI to Avicel was also significantly influenced by the length of drying time. To retain maximum sorption of PEI, it was necessary to dry the material below 7% moisture before washing with methanol. Removal of excess PEI was more efficient with absolute methanol than with water because of greater PEI solubility in the alcohol. Washing with methanol also contributes to fixation of polymer to the microcrystalline bead. When water was used for washing, leaching of PEI occurred during chromatography. Mixes containing more than 37% PEI were unstable when prepared as above. As indicated in Figure 2, acid-binding capacity was used as a criterion for measuring ion-exchange sites of the prepared batches of 1 and 3% PEI-Avicel (curves C and D, respectively). To obtain the curves, PEI-
CHROMATOGRAPHY
39
OF NUCLEOTIDES
Avicel (40 mg) in 5 ml of H,O was titrated to pH 12.0 with 0.5 1M NaOH and titrated back to pH 3.0 with 0.12 M HCl. Avicel was run also as a control to establish the amount of excess OH- added at pH 12 and also to compensate for the a’mount of acid that will bind with Avicel alone (curve L4). The milliequivalents of acid was then subtracted from curve A to determine the number of ion-exchange sites available. For example, the ion-exchange capacity of the preparation illustrated in curve C was 0.8 meq/gm in excess of that of the Avicel alone. The 3.0 5 2.5\” 5 2.0 z” f g 1.5d I 1.0 -
0/ 0/ p/
Z E 0.5 II
FIG. 1. Sorption Mixture was dried determination.
I 1.0
I 1.5
I 2.0 PEI, %
of PEI by Avicel concentrations for 96 hr and washed extensively
I 2.5
I 3.0
of PEI mixed with Avicel. with methanol before nitrogen
nitrogen content of the same preparation was 1.0 mmole/gm (Fig. 1). Approximately 80% of the PEI nitrogen that adhered to Avicel was available for ion exchange. The ion-exchange capacity of this PEIAvicel was considerably higher than that of the sample of commercial PEI-cellulose tested (curve B) . Selection of Gradient El&ion of Standard
System and Chromatography Nucleo tides
Figure 3 illustrates the influence of pH in maintaining peak height during chromatography and the influence of borate ion complexing on the elution position of nucleotides. The common 5’-monophosphates and
40
CHRISTIANSON,
PAULIS,
AND
WALL
3’-monophosphate of uridine plus UDP-glucose and ADP-glucose were eluted from 0.9 X 100 cm columns of Avicel prepared with 1% solution of PET under conditions described. In chromatogram A at pH 7 the sharpness of the peaks was diminished as the elution progressed. Consequently, it was necessary to increase protonixation of the ion exchanger by eluting with 0.6M LiCl at pH 4.5 (chromatogram B). The elution position of nucleotides was retarded since the column was not pre-
I 0
I 1
I I 2 3 Acid, meq.
I 4
I
5
FIQ. 2. Titration curves of PEI-Avicel showing pH change vs. meq acid added per gram: (A) Avicel only, (B) commercial PEI-cellulose, (C) PEI-Avicel made with 1% PEI (used for chromatography), (D) PEI-Avicel made with 3% PEI.
equilibrated ; however, the peak heights were ‘maintained under these conditions. The advantage of protonization was incorporated in the system used in chromatogram C. A pH gradient obtained with boric acid maintains sharpness of the nucleotides eluted last. Note the change in position of the sugar nucleotides. The pronounced effect of borate ions on the anion-exchange behavior of sugar nucleotides has been described (25).
CHROMATOGRAPHY
OF
41
NUCLEOTIDES
The final gradient elution system selected for routine use is shown in Figure 4. To ensure borate complexing of sugar nucleotides, deoxyribonucleoside monophosphates, and ribonucleoside monophosphates; pH 6.5 to 7.0 was maintained throughout this area of elution. Wucleoside monophosphates were eluted as sharp peaks with this slight pH change. The pH was then decreased by further addition of boric acid to maintain sharp peaks in the elution area of the nucleoside diphosphates. With this elution system, good resolution of standard 5’-mono- and 5’-diphosphates of uridine, cytidine, adenosine, and guanosine was achieved (Fig. 5). The 3’-UMP was also included in the mixture to demonstrate separation
B. LiCI-HCI pH 4.5
UDPG
ADPG 3’4MP ’
CMP GMP
FIG. 3. Separation of mixture of nucleoside monophosphates to illustrate influence of pH on peak height and borate complexing on relative elution position. All nucleotides are the 5’-isomers with the exception of the monophosphate of uridine, which is the 3’-isomer. Chromatogram B was not preconditioned to pH 4.5 before chromatography. Column: PEI-Avicel made with 1% PEI, 0.9 x 100 cm, bead size 38 m$. Elution system as described: flow rate, 45 ml/hr; O.bl.0 pmole of each substance was applied on column.
of this analog. The sugar nucleotides of glucose, here exemplified by ADP-glucose and UDP-glucose, were also separated. Along with these sugar nucleotides, deoxyribonucleotides were resolved from their ribonucleotide analogs by the presence of borate in the system. This effect was described previously (26), Most of the nucleotides were well resolved in a small volume (20-50 ml). This system was most satisfactory for a survey of the nucleotides in immature maize. For resolution of poorly resolved areas, rechromatography of desalted peaks with different gradient systems has been successful.
42
CHRISTIANSON,
PAULIS,
AND
WALL
Evidence has been obtained by thin-layer chromatography that PEIAvicel having higher exchange capacities than those employed here gives better resolution of nucleoside monophosphates. Higher capacity PEI-Avicel may be advantageous for use in column chromatography when these compounds are of major interest. Nucleoside triphosphates can also be separated on these columns by extending the elution gradient. Degradation occurs, however, during the time necessary for their elution. Separate chromatography on shorter
7.0
1.6 tt
Volume, ml.
FIG. 4. Gradient elution system of LiCl and HBOa for separation of mono- and diphosphate nucleotides based on six chambers (350 ml each) of a nine-chambered Varigrad. Chambers 1 and 2 contained H,O; chambers 3, 4, 5, and 6 contained 0.50, 2.0, 2.0, 2.0 A4 concentrations of LiCl and 0.526, 0.132, 0.395, and 0.526 M concentrations of HJSO,, respectively. The pH of the system during chromatography is illustrated.
columns, where more rapid elution results, diminished this degradation. Purine and pyrimidine bases and nucleosides do not interfere with chromatography of nucleoside monophosphates on PEI-Avicel. Their secondary binding interactions with this cellulose appear to be minimal. They are eluted very early, in contrast to their behavior on polystyrene resins, where they elute in some cases in the vicinity of some nucleoside monophosphates.
CHROMATOGRAPHY
Chromatography
of Immature
OF
KUCLEOTIDES
43
Maize Grain Extracts
Figure 6 shows the application of this chromatographic procedure for the separation of free nucleotides isolated from immature maize grain (25 days after pollination). The acid-soluble nucleotides were chromatographed directly without preliminary purification of the extract. Direct analysis of the extracts reduces degradation or losses of the nucleotides, especially sugar nucleotides. Use of charcoal adsorption and desorption with alkaline systems, or more extensive liquid-liquid ether extractions
FIQ. 5. Ion-exchange separation of a mixture of 5’-mono- and diphosphates of adenosine, guanosine, uridine, and eytosine, and also including 3’-monophosphate of uridine ; and adenosine diphosphate (ADP)-glucose (Glu), uridine diphosphate (UDP)-glucose (Glu). Guanine and guanosine were also added to the mixture to illustrate their elution positions. Column : PEI-Avicel made with 1% PEI, 0.9 x 100 cm, bead size 38 p. Elution system: simultaneous gradients of LiCl and HB08 with variable pH as shown in Figure 4. Column was washed with 175 ml Hz0 before start of gradient. Only LiCl gradient illustrated in this figure. Flow rate 45 ml/hr; 0.5-1.0 FmoIe of each substance was applied on column.
or both, for purification of the extract is avoided. The ultravioletabsorbing contaminants which these techniques seek to eliminate interfere with the chromatography on polystyrene resins. We observed that nucleotides in extracts of maize grain were not well resolved from residual phenolic materials on polystyrene resins even after preli,minary partial purification. Most of these nonnucleotide ultraviolet-absorbing materials were not adsorbed to the PEI-Avicel. Separation of these contaminants from nucleotides was achieved on PEI-Avicel columns by their elimination in the initial water wash. As much as 70% of the total absorbance applied to the column can be eliminated by washing. The
44
CHRISTIANSON,
PAULIS,
AND
WALL
low-baseline ultraviolet absorbance achieved throughout the run demonstrates further that little ultraviolet absorbing material is smeared through the column during fractionation. Even in the extreme case of mature maize extracts which contain higher contents of nonnucleotide materials including phenolic substances, an extended period of pre1.00 -’
- 0.75 2 z 3 0.50 % Z 2e 0.20 -
0
100
500
1000
Effluent, ml.
FIG. 6. Separation of acid-soluble nucleotides of immature maize grain (25 days after pollination). Chromatographic conditions similar to those described in Figure 5. Peaks, 5, 7, 8, and 10 were identified as UPD-X, GDP-X, 5’UMP, and 5’AMP, respectively. See Table 1 for details of characterization.
li,minary washing of the column also resulted in low blank values with well resolved peaks. Four of the major peaks from this separation were desalted and their composition was investigated. Analysis of the substituents of these nucleotides were performed directly on the desalted fraction -without further purification. Table 1 gives the analytical data compiled for their TABLE 1 Chemical Analysis of Nucleotides Isolated from Immature Composition, ‘E?
filll&S
5 7 8 10
5.5 11.8 13.3 6.0
““:“,‘e”
Uridine Guanosine Uridine Adenosine
= 25 days after pollination. b Peaks from Figure 6.
Compound identified
UDP-X GDP-X 5’-UMP 5’-AMP
Maize Graina
moles/mole
Total phosphorus
Total nitrogen
Pentose
1.75 2.40 1.20 1.00
1.90 3.72 2.10 5.42
1.08 0.90 0.99 0.86
base Reducing sugar after hydrolysis
1.00 0.96
CHROMATOGRAPHY
OF NUCLEOTIDES
45
identification. The sugar substituents of the two sugar nucleotides are still under investigation. A more detailed study of the variety and changes of nucleotides in separate embryo and endosperm fractions during maturation is in progress. The nucleotide separations possible with this method should permit further study of the role of nucleotides in the biosynthesis of various carbohydrates in the developing seed. SUMMARY
A procedure has been developed for the quantitative chromatographic separation of free nucleotides isolated from maize grain extracts on columns of polyethyleneimine-microcrystalline cellulose. Use of a uniform microcrystalline cellulose provides sharper separations of nucleotides as compared with separations with standard fibrous celluloses. Mono- and diphosphate nucleotides and sugar nucleotides are eluted with a concave gradient elution system of LiCl and boric acid, in which ionic strength and pH are independently changed. Borate complexing facilitates both separation of sugar nucleotides from their related diphosphate nucleotides and separation of deoxyribo- and ribonucleotides. Phenolic materials and other plant constituents that interfere with the detection of nucleotides during chromatography are less readily adsorbed on PEI-cellulose. Use of PEI-cellulose, therefore, permits direct analysis of some plant extracts without extensive preliminary purification, which may result in degradation and loss of nucleotides. Application of the method is illustrated by the analysis of free nucleotides contained in immature ‘maize seeds. REFERENCES 1. COHN, 2.
3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
W. E., J. Am. Chem. Sot. 72, 1471 (1950). BERGKVIST, R., Acta Chem. Stand. 10, 1303 (1956). GINSBURG, V., STUMPF, P. K., AND HASSID, W. Z., J. Biol. Chem. 223, 977 (1956). BROWN, E. G., Biochem J. 85,633 (1962). STAEHELIN, M., PETERSON, E. A., .~ND SOBER, H. A., Arch. Biochem. Biophys. 85, 289 (1959). TOMLINSON, R. V., AND TENER, G. M., Biochemistry 2, 697 (1963). NILSSON, R., AND SJUNNESSON, M., Acta Chem. Stand. 15, 1017 (1961). DAVEY, C. L., Biochim. Biophys. Acta 61, 538 (1962). OCKERMAN, P. A., Biochim. Biophys. Acta 74,588 (1963). STAEHELIN, M., B&him. Biophys. Actu 49, 11 (1961). GNDERATH, K., Angew. Chem. 74, 780 (1962); Intern. Ed. 1, 553 (1962). CHRISTIANSON, D. D., SINCLAIR, H. B., AND PAULIS, J. W., Biochim. Biophys. Actu 121, 412 (1966). RANDERATH, K., Biochim. Biophys. Acta 81,852 (1962). PETERSON, E. A., AND SOBER, H. A., Anal. Chem. 31,857 (1959). BLUMSOM, N. L., AND B.4DDILEY, J., Biochem. J. 81, 114 (1961).
46 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
CHRISTIANSON,
PAULIS,
AND
WALL
PALADINI, A. C., AND LELOIR, L. F., Biochem. J. 51,426 (1952). RANDERATH, K., AND RANDERATH, E., J. Chromatog. 16, 111 (1964). PARK, J. T., AND JOHNSON, M. J., J. Biol. Chem. 181, 149 (1949). DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, Chem. 28, 350 (1956). MEJBAUM, W., 2. Physiol. Chem. 258, 117 (1939). HURST, R. O., Can. J. Biochem. 42,287 (1964). DANKERT, M., PASSERON, S., RECONDO, E., AND LELOIR, L. F., Biochem. Res. Commun. 14, 1358 (1964). VISCHER, E., AND CHARGAFF, E., J. Biol. Chem. 186, 715 (1948). MIDDLETON, R. R., J. Appl. Chem. 10,281 (1960). RANDERATH, K., AND RANDERATH, E., Anal. Biochem. 13,575 (1965). KHYM, J. X., AND COHN, W. E., Biochim. Biophys. Acta 15, 139 (1954).
F., Anal.
Biophys.