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Separation of deoxyribonucleotides from ribonucleotides by anion-exchange thin-layer chromatography
622
BIOCHIMICA ET BIOPHYSICA ACTA
Short Communications SC 7101
Separation of deoxyribonucleotides from ribonucleotides by onion-exchange thin-layer chromatography Deoxyribonucleoside monophosphates can be separated from ribonucleoside monophosphate~ by paper electrophoresis 1, paper chromatography 2, anion-exchange column chromatography 3, and cation-exchange column chromatography z,4. None of these methods is, however, rapid or can be used if very small amounts (I/,g or less) are to be analyzed. Methods for the analytical separation of deoxyribonucleoside di- and triphosphates from their ribonucleoside analogues have not yet been described in the literature. Such methods might be useful in studies concerning the enzymic reduction of ribonucleotides to deoxyribonucleotides (c/. refs. 4-6) or the incorporation of ribonucleotides into DNA (c[. ref. 7)We wish to describe in this communication a rapid and sensitive method for separating deoxyribonucleotides from ribonucleotides. The compounds are chromatographed on thin layers of the anion-exchanger poly(ethyleneimine)-celluloses'9 with solutions of LiC1 in aqueous boric acid. The pronounced effect of borate ions on the anion-exchange behavior of cis-glycols in generaP ° and ribonucleoside monaphosphates in particularS, n is well known. Borate complexing adds a net negative charge to these compounds and thus increases their distribution coefficients on anion-exchangers. Experimental. A suspension of 15 g unmodified cellulose powder in ioo ml dialyzed 1 % poly(ethyleneimine) hydrochloride solution* is coated on degreased glass plates as described earlier 8& The plates are given a preliminary development with distilled water before chromatography 8& The principles of anion-exchange thin-layer chromatography have been described elsewhere 9. Solvents used: I. 2 % boric acid-2 M LiC1 (2 : I, v/v), for the separation of nucleoside monophosphates. 2. 4 % boric acid-4 M LiC1 (4:3, v/v), for the separation ol nucleoside triphosphates. Results. As can be seen from Figs. I and 2, a complete separation of deoxyribonucleotides from their ribonucleotide analogues containing the same base is obtained with the solvents mentioned. If boric acid is omitted, the RF values of deoxyribo- and ribonucleotides of the same type are identical. The rate of migration is also influenced by the bases; it decreases in the order thymidine (uridine) > cytidine 2> adenosine 2> guanosine derivatives. The similarity of the two chromatograms (Figs. I and 2) seems to be particularly noteworthy. The nucleoside diphosphates (not investigated) should behave similarly. The method is very sensitive: about O.l-O. 3 m/~mole (o.o3-0.15 #g) of each substance can be detected by examining the chromatograms under a short-wave ultraviolet lamp in a completely darkened room. The sensitivity can be considerably * A 5 ° % solution of p o l y ( e t h y l e n e i m i n e ) (free base) in w a t e r was o b t a i n e d f r o m t h e C h e m i r a d Corp., E a s t Brunswick, N . J . (U.S.A.).
Biochi~n. Biophvs. Acta, 76 (1963) 622-624
623
SHORT COMMUNICATIONS •-.- FRONT
O0 oe
oo OO ! O oo° o
ll
O
. . . . . . . . . . . . . O 1 234 5 ' 6 7 8 91011 121314
,---- START
I I IIIIIl F i g . I. T h i n - l a y e r [ c h r o m a t o g r a m of nucleoside 5"-monophosphates plus ADP plus ATP (2-3 mp m o l e s o f e a c h c o m p o u n d ) . 0. 5 m m t h i c k p o l y ( e t h y l e n e i m i n e ) - c e l h i l o s e layer. Solvent i. Developm e n t d i s t a n c e : IO.9 c m i n 45 r a i n . I = A M P ; 2 = d e A M P ; 3 = A M P + d e A M P ; 4 = GMP; 5 = deGMP; 6 = GMP+deGMP; 7 = CMP; 8 = deCMP; 9 = CMP+deCMP; io = UMP; ii = TMP; i2~= UMP+TMP; 13 = A D P ; 14 = A T E T h e s l i g h t e l o n g a t i o n o f t h e r i b o n u c l e o t i d e spots might be due to partial dissociation of the borate complexes.
FRONT
0O
oo O oo O0 O 0 oooO
O0 START
I111 IIIIII1[ L F i g , 2. T h i n - l a y e r c h r o m a t o g r a m Qf n u c l e o s i d e t r i p h o s p h a t e s p l u s A D E ( 2 - 3 m # m o l e s o f e a c h c o m p o u n d ) , o. 5 n u n t h i c k p o l y ( e t h y l e n e i m i n e ) - c e l l u l o s e l a y e r . S o l v e n t 2. D e v e l o p m e n t d i s t a n c e : lO. 5 c m i n 45 r a i n . I = A T P ; 2 = d e A T P ; 3 = A T P + d e A T P ; 4 = GTP; 5 = deGTP; 6 = GTP+ deGTP; 7 = CTP; 8 = deCTP; 9 = CTP+deCTP; xo = U T P ; I I = T T P ; I 2 = U T P + T T P ; 13 = A D P .
Biochira. Biophys. Acta, 7 6 ( I 9 6 3 ) 6 2 2 - - 6 2 4
62 4
SHORT COMMUNICATIONS
increased if labeled compounds are used and the detection is carried out by autoradiography. I wish to thank Dr. P. C. ZAMECNIK and Dr. H. M. KALCKARfor their generous support during the course of this work. This investigation was supported by grant 3 TE CA 5o18 from the U.S. Public Health Service. This is publication No. 114o of the Cancer Commission of Harvard University.
The John Collins Warren Laboratories O~ the Huntington Memorial Hospital o/ Harvard University, Massachusetts General Hospital, Boston, Mass., U.S.A.
KURT RANDERATH
Naturwissenscha[ten, 39 (1952) 86. p. REICHARD, Acta Chem. Scand., 12 (1958) 2048. J. X. KHYM AND W. E. COHN, Biochim. Biophys. dcta, 15 (1954) 139. p. REICHARD, A. ]~ALDESTEN AND L. I~UTBERG, J. Biol. Chem., 236 (1961) 115o. p. REICHARD, J. Biol. Chem., 237 (1962) 3513. S. S. COHEN, H. D. BARNER AND J. LICHTENSTEIN, J. Biol. Chem., 236 (1961) 1448. H, G. KLEMPERER, J. S. KRAKOW A.ND E. S. CANELLAKIS, Biochim. Biophys. Acta, 61 (1962) 43. K. I~ANDERATH,Biochim. Biophys. Acta, 61 (1962) 852. it K. t{.ANDERATH, Thin-layer Chromatography, Verlag C h e m i e VVeinheim a n d e r B e r g s t r a s s e , a n d A c a d e m i c Press, N e w York, 1963 . 10 j . X. KHYM, L. P. ZILL AND W. E. COHN,in C. CALMON AND T. R. E. I'{RESSMAN, Ion-Exchangers in Organic and Biochemistry, Interscience, N e w York, 1957, p. 392. 11 W. E. COHN AND V. J. ]3OLLUM, Biochim. Biophys. Acta, 48 (1961) 588. z L. JAENICKE AND I. VOLLBRECHTSHAUSEN,
z it 4 5 e '/ 8
Received July ISt, 1963 Biochim. Biophys. Acta, 76 (1963) 622-624 SC 7114
Froctionotion of D N A I. Countercurrent distribution of notive ond degraded D N A Heterogeneity of DNA molecules from a given tissue has been examined by a number of methods including ultracentrifuga] sedimentation analysis and helix-coil transition during thermal denaturation 1, and ion-exchange chromatography *. 1RNA has been fractionated by countercurrent distribution in a number of 2-phase systems according to base composition3, 4 and countercurrent distribution has also been applied to the fractionation of soluble RNA's 5. Preliminary studies e with countercurrent distribution of degraded commercial I)NA from calf thymus achieved fractionation dependent upon base composition but undenatured I)NA remained largely in the aqueous phase and was not fractionated by these methods. In the present studies it has been possible to fractionate DNA's from various sources by countercurrent distribution. I)NA was prepared from rat liver, Drosophila melanogaster and Escherichia coli by modifications of the 4-aminosalicylate-phenol method 7. Commercial I)NA from salmon sperm (Mann, "highly polymerized") was reprecipitated from aqueous solution with ethoxyethanol in the presence of 4 % lithium acetate. Degradation was achieved enzymically in some experiments by treating a 0.2 % solution of DNA Biochim. Biophys. Acta, 76 (1963) 624 627
BIOCHIMICA ET BIOPHYSICA ACTA
Short Communications SC 7101
Separation of deoxyribonucleotides from ribonucleotides by onion-exchange thin-layer chromatography Deoxyribonucleoside monophosphates can be separated from ribonucleoside monophosphate~ by paper electrophoresis 1, paper chromatography 2, anion-exchange column chromatography 3, and cation-exchange column chromatography z,4. None of these methods is, however, rapid or can be used if very small amounts (I/,g or less) are to be analyzed. Methods for the analytical separation of deoxyribonucleoside di- and triphosphates from their ribonucleoside analogues have not yet been described in the literature. Such methods might be useful in studies concerning the enzymic reduction of ribonucleotides to deoxyribonucleotides (c/. refs. 4-6) or the incorporation of ribonucleotides into DNA (c[. ref. 7)We wish to describe in this communication a rapid and sensitive method for separating deoxyribonucleotides from ribonucleotides. The compounds are chromatographed on thin layers of the anion-exchanger poly(ethyleneimine)-celluloses'9 with solutions of LiC1 in aqueous boric acid. The pronounced effect of borate ions on the anion-exchange behavior of cis-glycols in generaP ° and ribonucleoside monaphosphates in particularS, n is well known. Borate complexing adds a net negative charge to these compounds and thus increases their distribution coefficients on anion-exchangers. Experimental. A suspension of 15 g unmodified cellulose powder in ioo ml dialyzed 1 % poly(ethyleneimine) hydrochloride solution* is coated on degreased glass plates as described earlier 8& The plates are given a preliminary development with distilled water before chromatography 8& The principles of anion-exchange thin-layer chromatography have been described elsewhere 9. Solvents used: I. 2 % boric acid-2 M LiC1 (2 : I, v/v), for the separation of nucleoside monophosphates. 2. 4 % boric acid-4 M LiC1 (4:3, v/v), for the separation ol nucleoside triphosphates. Results. As can be seen from Figs. I and 2, a complete separation of deoxyribonucleotides from their ribonucleotide analogues containing the same base is obtained with the solvents mentioned. If boric acid is omitted, the RF values of deoxyribo- and ribonucleotides of the same type are identical. The rate of migration is also influenced by the bases; it decreases in the order thymidine (uridine) > cytidine 2> adenosine 2> guanosine derivatives. The similarity of the two chromatograms (Figs. I and 2) seems to be particularly noteworthy. The nucleoside diphosphates (not investigated) should behave similarly. The method is very sensitive: about O.l-O. 3 m/~mole (o.o3-0.15 #g) of each substance can be detected by examining the chromatograms under a short-wave ultraviolet lamp in a completely darkened room. The sensitivity can be considerably * A 5 ° % solution of p o l y ( e t h y l e n e i m i n e ) (free base) in w a t e r was o b t a i n e d f r o m t h e C h e m i r a d Corp., E a s t Brunswick, N . J . (U.S.A.).
Biochi~n. Biophvs. Acta, 76 (1963) 622-624
623
SHORT COMMUNICATIONS •-.- FRONT
O0 oe
oo OO ! O oo° o
ll
O
. . . . . . . . . . . . . O 1 234 5 ' 6 7 8 91011 121314
,---- START
I I IIIIIl F i g . I. T h i n - l a y e r [ c h r o m a t o g r a m of nucleoside 5"-monophosphates plus ADP plus ATP (2-3 mp m o l e s o f e a c h c o m p o u n d ) . 0. 5 m m t h i c k p o l y ( e t h y l e n e i m i n e ) - c e l h i l o s e layer. Solvent i. Developm e n t d i s t a n c e : IO.9 c m i n 45 r a i n . I = A M P ; 2 = d e A M P ; 3 = A M P + d e A M P ; 4 = GMP; 5 = deGMP; 6 = GMP+deGMP; 7 = CMP; 8 = deCMP; 9 = CMP+deCMP; io = UMP; ii = TMP; i2~= UMP+TMP; 13 = A D P ; 14 = A T E T h e s l i g h t e l o n g a t i o n o f t h e r i b o n u c l e o t i d e spots might be due to partial dissociation of the borate complexes.
FRONT
0O
oo O oo O0 O 0 oooO
O0 START
I111 IIIIII1[ L F i g , 2. T h i n - l a y e r c h r o m a t o g r a m Qf n u c l e o s i d e t r i p h o s p h a t e s p l u s A D E ( 2 - 3 m # m o l e s o f e a c h c o m p o u n d ) , o. 5 n u n t h i c k p o l y ( e t h y l e n e i m i n e ) - c e l l u l o s e l a y e r . S o l v e n t 2. D e v e l o p m e n t d i s t a n c e : lO. 5 c m i n 45 r a i n . I = A T P ; 2 = d e A T P ; 3 = A T P + d e A T P ; 4 = GTP; 5 = deGTP; 6 = GTP+ deGTP; 7 = CTP; 8 = deCTP; 9 = CTP+deCTP; xo = U T P ; I I = T T P ; I 2 = U T P + T T P ; 13 = A D P .
Biochira. Biophys. Acta, 7 6 ( I 9 6 3 ) 6 2 2 - - 6 2 4
62 4
SHORT COMMUNICATIONS
increased if labeled compounds are used and the detection is carried out by autoradiography. I wish to thank Dr. P. C. ZAMECNIK and Dr. H. M. KALCKARfor their generous support during the course of this work. This investigation was supported by grant 3 TE CA 5o18 from the U.S. Public Health Service. This is publication No. 114o of the Cancer Commission of Harvard University.
The John Collins Warren Laboratories O~ the Huntington Memorial Hospital o/ Harvard University, Massachusetts General Hospital, Boston, Mass., U.S.A.
KURT RANDERATH
Naturwissenscha[ten, 39 (1952) 86. p. REICHARD, Acta Chem. Scand., 12 (1958) 2048. J. X. KHYM AND W. E. COHN, Biochim. Biophys. dcta, 15 (1954) 139. p. REICHARD, A. ]~ALDESTEN AND L. I~UTBERG, J. Biol. Chem., 236 (1961) 115o. p. REICHARD, J. Biol. Chem., 237 (1962) 3513. S. S. COHEN, H. D. BARNER AND J. LICHTENSTEIN, J. Biol. Chem., 236 (1961) 1448. H, G. KLEMPERER, J. S. KRAKOW A.ND E. S. CANELLAKIS, Biochim. Biophys. Acta, 61 (1962) 43. K. I~ANDERATH,Biochim. Biophys. Acta, 61 (1962) 852. it K. t{.ANDERATH, Thin-layer Chromatography, Verlag C h e m i e VVeinheim a n d e r B e r g s t r a s s e , a n d A c a d e m i c Press, N e w York, 1963 . 10 j . X. KHYM, L. P. ZILL AND W. E. COHN,in C. CALMON AND T. R. E. I'{RESSMAN, Ion-Exchangers in Organic and Biochemistry, Interscience, N e w York, 1957, p. 392. 11 W. E. COHN AND V. J. ]3OLLUM, Biochim. Biophys. Acta, 48 (1961) 588. z L. JAENICKE AND I. VOLLBRECHTSHAUSEN,
z it 4 5 e '/ 8
Received July ISt, 1963 Biochim. Biophys. Acta, 76 (1963) 622-624 SC 7114
Froctionotion of D N A I. Countercurrent distribution of notive ond degraded D N A Heterogeneity of DNA molecules from a given tissue has been examined by a number of methods including ultracentrifuga] sedimentation analysis and helix-coil transition during thermal denaturation 1, and ion-exchange chromatography *. 1RNA has been fractionated by countercurrent distribution in a number of 2-phase systems according to base composition3, 4 and countercurrent distribution has also been applied to the fractionation of soluble RNA's 5. Preliminary studies e with countercurrent distribution of degraded commercial I)NA from calf thymus achieved fractionation dependent upon base composition but undenatured I)NA remained largely in the aqueous phase and was not fractionated by these methods. In the present studies it has been possible to fractionate DNA's from various sources by countercurrent distribution. I)NA was prepared from rat liver, Drosophila melanogaster and Escherichia coli by modifications of the 4-aminosalicylate-phenol method 7. Commercial I)NA from salmon sperm (Mann, "highly polymerized") was reprecipitated from aqueous solution with ethoxyethanol in the presence of 4 % lithium acetate. Degradation was achieved enzymically in some experiments by treating a 0.2 % solution of DNA Biochim. Biophys. Acta, 76 (1963) 624 627