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A method for the improved separation of soluble nucleotides by ion-exchange chromatography
SHORT COMMUNICATIONS
147
BBA SC 7037 A method for the improved seporotion of soluble nucleotides by ion-exchonge chromatography The relative importance of nucleotides and nucleic acids in cellular metabolism has resulted in the separation of soluble nucleotides b y ion-exchange chromatography becoming a routine laboratory practice. Despite the quantity and variety of nulceotide separations which have appeared in the literature, none appear to combine all the characteristics favourable for a good separation. The complexity of the mixture of nucleotides and nucleotide derivatives extractable from most tissues indicated that constant-molarity elution from an ion-exchange resin would not give sufficient resolution, and the concept of gradient elution was first applied to the separation of these compounds b y HURLBERT et al. z Their 15 × 1.8 cm column of Dowex-i X I o resin, with formate as the exchangeable ion, and a four-stage convex elution gradient of formic acid and formic acid containing formate, resulted in an incomplete separation; each peak, or group of peaks, was subsequently subjected to rechromatography with ammonium formate elution. A complete separation seemed improbable to them since they wrote, "the acid-solub.~e fraction of tissues contains such a complex mixture of compounds that complete resolution by any single column is unlikely". Many workers have since modified this method of separation. BERGKVIST 2 used the same type of resin and convex elution, but b y increasing the length of the column to 4o cm and introducing 5 additional buffers in the elution sequence, he was able to separate the majority of the nucleotides in the extracts of cereal plants. The total eluate collected from such a run was, however, more than 2o 1, most of the individual peaks requiring more than I 1 for elution. The modification of GILBERT AND YEMMa reduced the elution volume to a more suitable level, 1-5 1, and also reduced the number of buffers used to 5. Reduction of the gradient-elution mixing-volume at appropriate stages in the separation was responsible for the increased rate of elution of the nucleotides from the column and for the sharper peaks obtained. The convex nature of the gradient used, however, tended to crowd the initial peaks at each stage of elution, resulting in incomplete resolution. In 1958, PONIIS AND BLUMSOM6 introduced a new method of separation, with a very striking improvement in the resolution. They achieved this b y using a lower cross-linkage resin and a concave elution gradient, although the gradient, produced b y using two parallel-sided flasks, became really concave only for the final 200/0 of the run. The use of C1- as the exchangeable ion, with elution by CaC12 in dil. HC1, necessitated the recovery of the nucleotides as the insoluble calcium salts from an alcohol-ether mixture, rather than by the direct removal of formic acid and formate buifers by lyophilisation when the resin was used in the formate form. Moreover, the length of the column--I2O c m - necessitated the use of pressure in the manipulation of the separation, and more than 600 fractions, with a total volume of 7 1, were collected. A consideration of the theory of ion-exchange chromatography enables the prediction of improved resolution with the use of uniformly sized particles of a lower cross-linked resin. Since the rate of equilibration between ions and resin is dependent on the size of the resin particle, the use of uniformly sized particles results in a constant rate of exchange throughout the resin column, and hence the ions will move down the column with minimum diffusion. The degree of cross-linkage of the resin determines t?iochim. Biophys..4cta, 61 (1962) 147-149
148
SHORT COMMUNICATIONS
the space available for holding the exchanged ions within the resin. Consequently, for the efficient exchange of relatively large ions, such as nucleotides, a resin of low cross-linkage is required. The benefit of using a concave gradient, so elegantly demonstrated by the PONTIS AND BLUMSON separation, can also be predicted on theoretical grounds s. Although the gradient produced by using parallel-sided vessels for reservoir and mixing flask is mathematically concave, the initial 8o °/o of the gradient is practically linear, and the full benefits of a truly concave gradient cannot be obtained. The replacement of the parallel-sided reservoir b y a conical flask produces a really concave gradient, very gently sloped initially but very steep at the end. The degree of concavity of the gradient can be controlled b y the shape and size of the conical reservoir relative to the mixing flask. The importance of column dimensions in affecting a separation is often overlooked or grossly underestimated. When a gradientelution system is being employed, increase of column length does not necessarily improve resolution 5 and consequently much shorter columns can often be used with no reduction in resolving power. Similarly, the best column diameter to give m a x i m u m resolution is not always sought. A small reduction in column width will usually allow the elution of a peak in a much smaller volume of eluent, and hence give better resolution. Consideration of these factors suggested the optimum conditions which might be used for the separation of nucleotides b y ion-exchange chromatography. The resin chosen was Dowex-I X4, 2o0-4o0 mesh, a quaternary ammonium polystyrene resin of relatively low cross-linkage, and this was further graded 6 to give a fraction of which 90% of the particles were within the diameter range 35-75/1- The resin was used in the formate form, and the elution of the nucleotides, exchanged onto the resin at p H 7, was brought about in two stages_ (I) Elution by increasing the formic acid concentration to 4 N, i.e. a p H change from 7-1.3, with a low formate ion concentration, and (2) elution by increasing the formate ion concentration to I M, the acid concentration being kept constant at 4 N. Concave gradients were used for both stages. For the first stage of elution the mixing flask (parallel-sided, 9.5 cm diameter) contained 550 ml of water, and IO N formic acid was fed into this from the reservoir (5oo-ml conical flask containing 475 ml acid) until a concentration of 4 N acid was obtained. (The two flasks were positioned such that the levels of the liquids were equal, and a capillary siphon connection was made between them. The rates of movement of liquid into and out of the mixing flask, which determine the shape of the gradient produced, are directly dependent on the cross-sectional areas of the two flasks. The area of the reservoir changes continuously throughout the run, the diameter increasing from 4-5 to 8.5 cm) The volumes of water and acid used were such that 17 ° fractions, each of 3.2 ml, were collected during this time. The second gradient was obtained by filling up the mixing flask with 4 N formic acid (550 ml), and siphoning in 1.6 M ammonium formate in 4 N acid (475 ml in reservoir) for a further 230 fractions, b y which time the mixing flask contained i.o M formate in 4 N acid (Fig. I). This concentration was sufficient to elute all the nucleotides from the resin. Using a 30 × I.O cm column a convenient flow rate of 15-2o ml/h was obtained without the use of pressure, The separation of nucleotides achieved by this method is apparent from Fig. I, which shows the analysis of an ethanolic extract of the yeast, Torulopsis utilis. Identification of the peaks was b y their ultraviolet spectral data, b y co-chromatography with standard samples, by paper chromatography in two solvents and b y hydrolysis Biochim. Biophys. Acta, 61 (1962) I 4 7 - 1 4 9
x49
SHORT COMMUNICATIONS
I
AIP
OPN~ AMP
f
UOP-PepLlde
/
E E 05M
OE
•
•
~
P
E
.< 10
E ,,(
~-" 4N
~/ ~
G~P
ODP
. ~ GMP
2N
7~
,too FracLton number(B2mt)
total
volume 13 I
24
7
/
Fig. i. The s e p a r a t i o n of the nucleotides from a 6 o % - E t h a n o ] E x t r a c t of Torulopsis utiIis. A.3o × i.o cm c o l u m n of D o w e x - i X4, f o r m a t e resin, 2oo-4oo m e s h graded to 3 5 - 7 5 1 ~ was used. The b r o k e n line indicates the g r a d i e n t elution used.
to the constituent base. In addition to the common nucleotides, several peaks containing more complex compounds were observed. Peak I contained all those compounds extracted by the ethanol which have no negative charge at pH 7, such as the bulk of the amino acids and sugars. Peaks 3 and 4 appeared to contain cytidine phosphate in a conjugated form, and peaks 2, 5, 6, 7 and 8 all appeared to be derivatives of adenosine phosphate. This method of separation has successfully been used in an investigation of the metabolism of nucleotides and nucleic acids in exponentially growing T. ~ti2is, and also to follow the changes in nucleotide composition and content which accompany the germination of barley grain (results to be published elsewhere). This separation, developed from a critical consideration of the/actors influencing an ion-exchange separation, gives good resolution of a relatively complex soluble nueleotide extract, coupled with easy recovery of the nucleotides, convenience in manipulation, and the use of a minimal volume of eluate. The author is indebted to Prof. E, W. YEMM, and Drs. B. F. FoL~ts and A. P. SI~S for helpful discussion during the course of the work, and to D. S. I. R. for the Research Grant.
Depa,rtment o[ Botany, U~iversity o/ Bristol, Bristol (Great Britain)
JOHN INGLE
1 19,. t3, tlURImEgT, H. SCHMITZ, A. F. BRUMM AND V. R. POa'TER, J. Biol. Chem., 209 (I954) 73. R. Bt~RGKVIST, Acta Chem. ScamJ., Io (t955) I3o3 . "~ D. A. GILI4ER'r A~D E. "~V. Y ~ g , iVature, I8~ (I958) I745. a H. ~. PONTIS AND N. L. [:~LU'MSOM, Biochim_ Biophys. ,,Icta, 27 (t958) 6I$. T. K. LAKSHMANAN AND S. LIEBER~tX~, Arch. Biochem. Biophys., 53 (I954) 258. s p_ 13. HAMILTON, ,'(nag. Chem., 3 ° (i958) 914 .
Received April 9th, 1962. " P r e s e n t Address: D e p a r t m e n t of A g r o n o m y , University o[ Illinois, Urbana, I11. {U.S.A.)
Bioehir~. B i o p h y s . Acta. 6z (1962) I 4 7 - 1 4 9
147
BBA SC 7037 A method for the improved seporotion of soluble nucleotides by ion-exchonge chromatography The relative importance of nucleotides and nucleic acids in cellular metabolism has resulted in the separation of soluble nucleotides b y ion-exchange chromatography becoming a routine laboratory practice. Despite the quantity and variety of nulceotide separations which have appeared in the literature, none appear to combine all the characteristics favourable for a good separation. The complexity of the mixture of nucleotides and nucleotide derivatives extractable from most tissues indicated that constant-molarity elution from an ion-exchange resin would not give sufficient resolution, and the concept of gradient elution was first applied to the separation of these compounds b y HURLBERT et al. z Their 15 × 1.8 cm column of Dowex-i X I o resin, with formate as the exchangeable ion, and a four-stage convex elution gradient of formic acid and formic acid containing formate, resulted in an incomplete separation; each peak, or group of peaks, was subsequently subjected to rechromatography with ammonium formate elution. A complete separation seemed improbable to them since they wrote, "the acid-solub.~e fraction of tissues contains such a complex mixture of compounds that complete resolution by any single column is unlikely". Many workers have since modified this method of separation. BERGKVIST 2 used the same type of resin and convex elution, but b y increasing the length of the column to 4o cm and introducing 5 additional buffers in the elution sequence, he was able to separate the majority of the nucleotides in the extracts of cereal plants. The total eluate collected from such a run was, however, more than 2o 1, most of the individual peaks requiring more than I 1 for elution. The modification of GILBERT AND YEMMa reduced the elution volume to a more suitable level, 1-5 1, and also reduced the number of buffers used to 5. Reduction of the gradient-elution mixing-volume at appropriate stages in the separation was responsible for the increased rate of elution of the nucleotides from the column and for the sharper peaks obtained. The convex nature of the gradient used, however, tended to crowd the initial peaks at each stage of elution, resulting in incomplete resolution. In 1958, PONIIS AND BLUMSOM6 introduced a new method of separation, with a very striking improvement in the resolution. They achieved this b y using a lower cross-linkage resin and a concave elution gradient, although the gradient, produced b y using two parallel-sided flasks, became really concave only for the final 200/0 of the run. The use of C1- as the exchangeable ion, with elution by CaC12 in dil. HC1, necessitated the recovery of the nucleotides as the insoluble calcium salts from an alcohol-ether mixture, rather than by the direct removal of formic acid and formate buifers by lyophilisation when the resin was used in the formate form. Moreover, the length of the column--I2O c m - necessitated the use of pressure in the manipulation of the separation, and more than 600 fractions, with a total volume of 7 1, were collected. A consideration of the theory of ion-exchange chromatography enables the prediction of improved resolution with the use of uniformly sized particles of a lower cross-linked resin. Since the rate of equilibration between ions and resin is dependent on the size of the resin particle, the use of uniformly sized particles results in a constant rate of exchange throughout the resin column, and hence the ions will move down the column with minimum diffusion. The degree of cross-linkage of the resin determines t?iochim. Biophys..4cta, 61 (1962) 147-149
148
SHORT COMMUNICATIONS
the space available for holding the exchanged ions within the resin. Consequently, for the efficient exchange of relatively large ions, such as nucleotides, a resin of low cross-linkage is required. The benefit of using a concave gradient, so elegantly demonstrated by the PONTIS AND BLUMSON separation, can also be predicted on theoretical grounds s. Although the gradient produced by using parallel-sided vessels for reservoir and mixing flask is mathematically concave, the initial 8o °/o of the gradient is practically linear, and the full benefits of a truly concave gradient cannot be obtained. The replacement of the parallel-sided reservoir b y a conical flask produces a really concave gradient, very gently sloped initially but very steep at the end. The degree of concavity of the gradient can be controlled b y the shape and size of the conical reservoir relative to the mixing flask. The importance of column dimensions in affecting a separation is often overlooked or grossly underestimated. When a gradientelution system is being employed, increase of column length does not necessarily improve resolution 5 and consequently much shorter columns can often be used with no reduction in resolving power. Similarly, the best column diameter to give m a x i m u m resolution is not always sought. A small reduction in column width will usually allow the elution of a peak in a much smaller volume of eluent, and hence give better resolution. Consideration of these factors suggested the optimum conditions which might be used for the separation of nucleotides b y ion-exchange chromatography. The resin chosen was Dowex-I X4, 2o0-4o0 mesh, a quaternary ammonium polystyrene resin of relatively low cross-linkage, and this was further graded 6 to give a fraction of which 90% of the particles were within the diameter range 35-75/1- The resin was used in the formate form, and the elution of the nucleotides, exchanged onto the resin at p H 7, was brought about in two stages_ (I) Elution by increasing the formic acid concentration to 4 N, i.e. a p H change from 7-1.3, with a low formate ion concentration, and (2) elution by increasing the formate ion concentration to I M, the acid concentration being kept constant at 4 N. Concave gradients were used for both stages. For the first stage of elution the mixing flask (parallel-sided, 9.5 cm diameter) contained 550 ml of water, and IO N formic acid was fed into this from the reservoir (5oo-ml conical flask containing 475 ml acid) until a concentration of 4 N acid was obtained. (The two flasks were positioned such that the levels of the liquids were equal, and a capillary siphon connection was made between them. The rates of movement of liquid into and out of the mixing flask, which determine the shape of the gradient produced, are directly dependent on the cross-sectional areas of the two flasks. The area of the reservoir changes continuously throughout the run, the diameter increasing from 4-5 to 8.5 cm) The volumes of water and acid used were such that 17 ° fractions, each of 3.2 ml, were collected during this time. The second gradient was obtained by filling up the mixing flask with 4 N formic acid (550 ml), and siphoning in 1.6 M ammonium formate in 4 N acid (475 ml in reservoir) for a further 230 fractions, b y which time the mixing flask contained i.o M formate in 4 N acid (Fig. I). This concentration was sufficient to elute all the nucleotides from the resin. Using a 30 × I.O cm column a convenient flow rate of 15-2o ml/h was obtained without the use of pressure, The separation of nucleotides achieved by this method is apparent from Fig. I, which shows the analysis of an ethanolic extract of the yeast, Torulopsis utilis. Identification of the peaks was b y their ultraviolet spectral data, b y co-chromatography with standard samples, by paper chromatography in two solvents and b y hydrolysis Biochim. Biophys. Acta, 61 (1962) I 4 7 - 1 4 9
x49
SHORT COMMUNICATIONS
I
AIP
OPN~ AMP
f
UOP-PepLlde
/
E E 05M
OE
•
•
~
P
E
.< 10
E ,,(
~-" 4N
~/ ~
G~P
ODP
. ~ GMP
2N
7~
,too FracLton number(B2mt)
total
volume 13 I
24
7
/
Fig. i. The s e p a r a t i o n of the nucleotides from a 6 o % - E t h a n o ] E x t r a c t of Torulopsis utiIis. A.3o × i.o cm c o l u m n of D o w e x - i X4, f o r m a t e resin, 2oo-4oo m e s h graded to 3 5 - 7 5 1 ~ was used. The b r o k e n line indicates the g r a d i e n t elution used.
to the constituent base. In addition to the common nucleotides, several peaks containing more complex compounds were observed. Peak I contained all those compounds extracted by the ethanol which have no negative charge at pH 7, such as the bulk of the amino acids and sugars. Peaks 3 and 4 appeared to contain cytidine phosphate in a conjugated form, and peaks 2, 5, 6, 7 and 8 all appeared to be derivatives of adenosine phosphate. This method of separation has successfully been used in an investigation of the metabolism of nucleotides and nucleic acids in exponentially growing T. ~ti2is, and also to follow the changes in nucleotide composition and content which accompany the germination of barley grain (results to be published elsewhere). This separation, developed from a critical consideration of the/actors influencing an ion-exchange separation, gives good resolution of a relatively complex soluble nueleotide extract, coupled with easy recovery of the nucleotides, convenience in manipulation, and the use of a minimal volume of eluate. The author is indebted to Prof. E, W. YEMM, and Drs. B. F. FoL~ts and A. P. SI~S for helpful discussion during the course of the work, and to D. S. I. R. for the Research Grant.
Depa,rtment o[ Botany, U~iversity o/ Bristol, Bristol (Great Britain)
JOHN INGLE
1 19,. t3, tlURImEgT, H. SCHMITZ, A. F. BRUMM AND V. R. POa'TER, J. Biol. Chem., 209 (I954) 73. R. Bt~RGKVIST, Acta Chem. ScamJ., Io (t955) I3o3 . "~ D. A. GILI4ER'r A~D E. "~V. Y ~ g , iVature, I8~ (I958) I745. a H. ~. PONTIS AND N. L. [:~LU'MSOM, Biochim_ Biophys. ,,Icta, 27 (t958) 6I$. T. K. LAKSHMANAN AND S. LIEBER~tX~, Arch. Biochem. Biophys., 53 (I954) 258. s p_ 13. HAMILTON, ,'(nag. Chem., 3 ° (i958) 914 .
Received April 9th, 1962. " P r e s e n t Address: D e p a r t m e n t of A g r o n o m y , University o[ Illinois, Urbana, I11. {U.S.A.)
Bioehir~. B i o p h y s . Acta. 6z (1962) I 4 7 - 1 4 9