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Rapid resolution of 5′-mono-, -di-, and -triphosphate ribo- and deoxyribonucleoside mixtures by conventional anion-exchange chromatography
Note
I. X. KHYM Biology
Di&ion,
Oak
R&&e National
Loboratory,
Oak
Ridge,
Term. 37830 (U.S.A.)
(Received March 15th. 1976)
Ever since chromatograljtiic techniques have heen applied to the task of separating nucleic acid components (for reviews, see refs. l-4), considerable thought and effort has been put forth, by investigators from many different laboratories, to solve the difficult problem of separating complex nucleotide mixtures. As a result of these efforts, several different chromatographic methodss-13 are now available for the rapid separation of complex mixtures of either ribo- or deoxyribomononucleoside phosphates, but only iimited success has been achieved in separating mixtures that contain all members of each class of nucleotide. For instance, simple mixtures of ribo- and deoxyribonucleoside monophosphates can be resolved from solutions containing borateI utilizing the techniques of paper’“, electrophoresis’*“, ion-exchange1*1’*15, or thin-iayer chromato,mphy (TEC)16. Randerath and co-worker@‘-” have refined the latter technique and have developed two-dimensional TLC procedures to separate more complicated mixtures containing ribo- and deoxyribonucleotides at either the mono-, or di-, or triphosphate levels. But combinations of ribo- and deoxyribonucleotides at mixed phosphorylated levels are only partially resolved by the TLC procedures. By refinements of the conventional ion-exchange method13 used in this laboratory to separate nucleotides”, the resolution of mixtures that contain ribo- and de-
oxyribo-S-nucleotides at the mono-, di- and triphosphate levels, OF any combination of these, has been accomplished. This can be seen by thechromatogram shown in Fig. 1. As noted in the legend to Fig. I, a gradient delay and a lower initial pH are used to effect the separation shown. The gradient delay allows the solute bands of closely eluting species (e.g., CD?, dCDP and GMP, OF CTP and dCTP) to become
somewhat separated on the column at low citrate concentration before the bands are removed from the column as sharp peaks by the increasing citrate concentrations of the gradient. The lower initial pH of 8.2 is n-sary to resolve the UMP-dTMP pair; it also seems to effect the resolution of dTDP from CFP. The change to pH S.6 (citrate concentration still at 25 mA4) after the first 20 rnin is necessary to effect the separation * Re supported by the Energy Researchuld Development Admkistxation tmdercontrzct with finion Carbide Corporation. = Bates 2nd nuckosides may be convenientlyremoved from nucleotidemixtures by polyacrylzmide chromatography”. l
L
25 TfME (h)
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-
._ .-
.I W; E. C&h& in E, Charg&Iznd J. N. Davidson (Editors), The Nrcclei Acids, Vol. I, Academic PI&F, New York:1955, p. 21t. 2 G. R_ Wyatt. in E_ &argatF and J. N. Davidsod (Editors), 2%~ Nucleic Acids, Vol. I, Academic PresS, New York. L955, p. 243. Vol. 1, Academic 3 J. D. Smith, in E. C&w&f and J. N. Davidson @ditors), The Nc~cfe~c AC& Press, New xx-k, 1955, p; 267. 4 J. I Saukkonen, CJITamzrtigr_ Rev., 26 (L964) 53. 5 EC_Randera&, ?Xi;-taver Chromatography, VerIag-Chemie, Weinheim,Bergstr. and Amdemic Press, New.York, I963. 6 K. Randemth uld E Randerath, L? L. Grossman and K. Moldave (Editors), Me~Iwds of Envymo&qy. Vol. XfIA, Academic Press, New York, 1967. p_ 323. 5 J. D. Smith, in k Grossman ud K. Moldave (Editors), Metfiods of Enzymufogy, Vol. XUA, Academic Ekes, New York, 1957, p_ 3150~ C. Horva&, MethaLF Bimfrenz. Ad., 21 (1973) 79. R Sa Sep. P&f. Methuds, 3 (1974) 339. P. M. ?Zajcsanyi M. Csil@g and E. Kliskovics, Sep. Purif. Metfwds, 3 (1974) 167. F. R- Brown, Higlt-pesure L&&d Chrmrzatography, lnterscience New York, 1973. R A.. Hartwick and P. R Brown, J. Cfzromatogr., 1 L2 (1975) 551. J; X. Khym, Ch. Chem., 21 (1975) 124.5. I. X. EChym, in L. Grossmar~ and K. MoIdave (Editors), .%fer~&s of Ehzynwlugy, Vol. XILA, Academic P_zss New York, 1967, p_ 93. J. X. Kkym and W. E. Cohn, Bkxhim. Brbphys .Acta, 1.5 (19% 139. K. Randerzth, Bio&im_ Biophyx Rcta, 76 (1963) 622_ #. Ra?derati and E. R&~ieraffi, J. Chromafogr., 1-6 (i964) 1 Il. E. Randenth and K. Randerath. J. Ckromafogr., 16 (19&?) 126.
9” 10
11 12 13 14
15 I6 17 18 19 J. Neuhard, E. Randersth and K. Randerath, ARK. Biochem., 20 J. X. Khym.
Anal. Eioclen~., 71 (1976) 231.
I3 (1965) 211.
I. X. KHYM Biology
Di&ion,
Oak
R&&e National
Loboratory,
Oak
Ridge,
Term. 37830 (U.S.A.)
(Received March 15th. 1976)
Ever since chromatograljtiic techniques have heen applied to the task of separating nucleic acid components (for reviews, see refs. l-4), considerable thought and effort has been put forth, by investigators from many different laboratories, to solve the difficult problem of separating complex nucleotide mixtures. As a result of these efforts, several different chromatographic methodss-13 are now available for the rapid separation of complex mixtures of either ribo- or deoxyribomononucleoside phosphates, but only iimited success has been achieved in separating mixtures that contain all members of each class of nucleotide. For instance, simple mixtures of ribo- and deoxyribonucleoside monophosphates can be resolved from solutions containing borateI utilizing the techniques of paper’“, electrophoresis’*“, ion-exchange1*1’*15, or thin-iayer chromato,mphy (TEC)16. Randerath and co-worker@‘-” have refined the latter technique and have developed two-dimensional TLC procedures to separate more complicated mixtures containing ribo- and deoxyribonucleotides at either the mono-, or di-, or triphosphate levels. But combinations of ribo- and deoxyribonucleotides at mixed phosphorylated levels are only partially resolved by the TLC procedures. By refinements of the conventional ion-exchange method13 used in this laboratory to separate nucleotides”, the resolution of mixtures that contain ribo- and de-
oxyribo-S-nucleotides at the mono-, di- and triphosphate levels, OF any combination of these, has been accomplished. This can be seen by thechromatogram shown in Fig. 1. As noted in the legend to Fig. I, a gradient delay and a lower initial pH are used to effect the separation shown. The gradient delay allows the solute bands of closely eluting species (e.g., CD?, dCDP and GMP, OF CTP and dCTP) to become
somewhat separated on the column at low citrate concentration before the bands are removed from the column as sharp peaks by the increasing citrate concentrations of the gradient. The lower initial pH of 8.2 is n-sary to resolve the UMP-dTMP pair; it also seems to effect the resolution of dTDP from CFP. The change to pH S.6 (citrate concentration still at 25 mA4) after the first 20 rnin is necessary to effect the separation * Re supported by the Energy Researchuld Development Admkistxation tmdercontrzct with finion Carbide Corporation. = Bates 2nd nuckosides may be convenientlyremoved from nucleotidemixtures by polyacrylzmide chromatography”. l
L
25 TfME (h)
-. -_
,_-
._--
.-.
-
._ .-
.I W; E. C&h& in E, Charg&Iznd J. N. Davidson (Editors), The Nrcclei Acids, Vol. I, Academic PI&F, New York:1955, p. 21t. 2 G. R_ Wyatt. in E_ &argatF and J. N. Davidsod (Editors), 2%~ Nucleic Acids, Vol. I, Academic PresS, New York. L955, p. 243. Vol. 1, Academic 3 J. D. Smith, in E. C&w&f and J. N. Davidson @ditors), The Nc~cfe~c AC& Press, New xx-k, 1955, p; 267. 4 J. I Saukkonen, CJITamzrtigr_ Rev., 26 (L964) 53. 5 EC_Randera&, ?Xi;-taver Chromatography, VerIag-Chemie, Weinheim,Bergstr. and Amdemic Press, New.York, I963. 6 K. Randemth uld E Randerath, L? L. Grossman and K. Moldave (Editors), Me~Iwds of Envymo&qy. Vol. XfIA, Academic Press, New York, 1967. p_ 323. 5 J. D. Smith, in k Grossman ud K. Moldave (Editors), Metfiods of Enzymufogy, Vol. XUA, Academic Ekes, New York, 1957, p_ 3150~ C. Horva&, MethaLF Bimfrenz. Ad., 21 (1973) 79. R Sa Sep. P&f. Methuds, 3 (1974) 339. P. M. ?Zajcsanyi M. Csil@g and E. Kliskovics, Sep. Purif. Metfwds, 3 (1974) 167. F. R- Brown, Higlt-pesure L&&d Chrmrzatography, lnterscience New York, 1973. R A.. Hartwick and P. R Brown, J. Cfzromatogr., 1 L2 (1975) 551. J; X. Khym, Ch. Chem., 21 (1975) 124.5. I. X. EChym, in L. Grossmar~ and K. MoIdave (Editors), .%fer~&s of Ehzynwlugy, Vol. XILA, Academic P_zss New York, 1967, p_ 93. J. X. Kkym and W. E. Cohn, Bkxhim. Brbphys .Acta, 1.5 (19% 139. K. Randerzth, Bio&im_ Biophyx Rcta, 76 (1963) 622_ #. Ra?derati and E. R&~ieraffi, J. Chromafogr., 1-6 (i964) 1 Il. E. Randenth and K. Randerath. J. Ckromafogr., 16 (19&?) 126.
9” 10
11 12 13 14
15 I6 17 18 19 J. Neuhard, E. Randersth and K. Randerath, ARK. Biochem., 20 J. X. Khym.
Anal. Eioclen~., 71 (1976) 231.
I3 (1965) 211.