Separation of nucleosides on Sephadex G-10

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BBA 93279

Separation of nucleosides on Sephadex G-IO We have previously reported on the usefulness of a new gel filtration material, Sephadex G-Io, for the separation of bases, nucleosides and nucleotides 1. Since the technique appeared particularly suitable for separating nucleosides, the method has been further improved to allow the separation of more complex mixtures of ribosides and deoxyribosides. Recently, other variants of the chromatographic separation of bases, nucleosides and nucleotides on columns of Sephadex G-io have been reported b y DIRHEIMER AND EBEL 2 a n d MEZZASOMA AND FARINA 3,4.

Materials were purchased as reported previously 1. 5-Iododeoxyuridine, pseudouridine and 5-hydroxymethyldeoxyuridine came from Calbiochem, Lucerne, Switzerland, and tritiated uridine from the Radiochemical Centre, Amersham, England. The chromatography apparatus consisted of a 90 cm x 1. 5 cm Sephadex column, an ultraviolet flow-through cell, an L K B Uvicord II recorder (254 m#) and an LKB fraction collector. Substances were applied in 0.5 ml water at a concentration of 0.2 mg/ml, giving o.I mg of each substance per chromatographic separation. The column was eluted at a flow rate of 25 ml/h, controlled by a peristaltic pump. Radioactivity was determined in a liquid scintillation counter. The separation of the 4 RNA ribosides under different conditions of elution is as follows. Under conditions similar to those used before z (phosphate buffer, pH 7.0) adenosine and guanosine separated well from each other and from the pyrimidine ribosides, but cytidine and uridine were hardly resolved. The sequence of elution was (Cyd+Urd), Guo, Ado. At pH 4.8 the same elution pattern was obtained. Eluting with formic acid at pH 2. 9, the profile was completely different: all substances eluted faster, in particular cytidine and adenosine. Cytidine and uridine were now well separated, but uridine and adenosine partly overlapped, the sequence being Cyd, Urd, Ado, Guo. Acetic acid at pH 3.3 only separated purine ribosides from pyrimidine ribosides, with the pyrimidine ribosides eluting first. An excellent separation was achieved by using a citric acid-phosphate buffer at pH 3-5 (Fig. I). Here all 4 nucleosides were well resolved. This system was checked by adding tritium-labelled uridine to the usual mixture of the 4 nucleosides. Radioactivity and absorbance of uridine coincide and it can be seen that trailing is minimal. The chromatographic yield was checked in a similar experiment and found to lie between 9° and IOO o~, based both on radioactivity and on absorbance. Separation of the 4 DNA deoxyribosides was also very good in the citric acidphosphate system. The elution pattern of deoxyribosides was the same as that of ribosides. Fig. 2 shows that thymidine is well separated from its analogs 5-bromodeoxyuridine and 5-iododeoxyuridine, but not from 5-fluorodeoxyuridine. The following substances were poorly resolved: 5-hydroxymethyldeoxyuridine from uridine, and pseudo-uridine from uridine. This method may be used to determine nucleic acid composition. In preliminary Biochim. Biophys. Acta, 149 (1967) 6Ol-6O3

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Fig. I. Separation om Sepha.dex G-IO of a m i x t u r e of the 4 R N A ribosides, to which 5/~C [aH]uridine had been added. The b u f f e r was o . o i M citric a c i d - N a 2 H P O 4 (pH 3.5). Fractions of 2.5 m l were collected; a b s o r b a n c e at 260 m/z w a s m e a s u r e d a n d aliquots were used for counting of radioactivity. The a b s o r p t i o n of t h e first 23 fractions w a s only checked on t h e a u t o m a t i c f l o w - t h r o u g h recorder. Peaks: i, dextralx blue; 2, cytidine; 3, uridine; 4, adenosine; 5, guanosine.

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Fig. 2. Separation of t h y m i d i n e and its analogs on S e p h a d e x G - i o using o.oi M citric a c i d - N a , H P O , b u f f e r (pH 3.5)- Peaks: i, d e x t r a n blue; 2, t h y m i d i n e + 5 - f l u o r o d e o x y u r i d i n e ; 3, 5 - b r o m o d e o x y uridine; 4, 5-iododeoxy uridine. The a m o u n t of t h y m i d i n e added w a s 5 times higher t h a n u s u a l

Biochim. Biophys. Acta, 149 (1967) 6Ol-6O 3

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experiments, RNA was subjected to alkaline hydrolysis; the resulting nucleotides were dephosphorylated by alkaline phosphatase and the nucleoside mixture was separated on Sephadex G-Io. At pH 3.5 nucleotides and nucleosides partly overlap, while they are well resolved at pH 7.0. In summary, by a suitable choice of elution media, columns of Sephadex G-Io can be used for the efficient separation of nucleosides. No gradients are required and the same column can be used many times without regeneration. A typical separation requires 8-1o h. The skilful help of Miss MARLISSCHUMACHERand Miss HELEN WlLI is gratefully acknowledged. Financial support was provided by the "Fonds National Suisse de la Recherche Scientifique".

Department o~ Cell Biology, Swiss Institute/or Experimental Cancer Research, Lausanne (Switzerland) I 2 3 4

RICHARD BRAUN

R. BRAUN, Biochim. Biophys. Acta, 142 (1967) 267. G. DIRHEIMER AND J. P. EBEL, Bull. Soc. Chim. Biol. 49 (1967) 447. I. 3{EZZASOMAAND B. FARINA, Boll. Soc. Ital. Biol. Sper., 42 (1966) 1449. I. 1ViEZZASOMAAND B. FARINA, Boll. SOC. Ital, Biol. Sper., 43 (1966) 29.

Received July 25th, 1967 Revised manuscript received October Ilth, 1967 Biochim. Biophys. Acta, 149 (1967) 6Ol-6O 3

BBA 93274 Studies of replicating D N A of regenerating rat liver using chemical modifications with water-soluble carbodiimide We have reported earlier that DNA isolated from regenerating rat liver during the S period ( 2 0 - 2 4 h after partial hepatectomy), in comparison with that isolated at the pre- or post-synthetic periods, exhibits some properties of the denatured state 1,*. About 15 % of this DNA, like denatured DNA, is removed into the interface by fractionating in two-phase system chloroform-water. The incorporation of [14C]thymidine in vivo into this denatured fraction is 6-1o times greater than into the native one. A number of workers have found some denatured DNA properties in fractions of DNA isolated from growing bacteria, human cell cultures and. Ehrlich ascitic carcinoma cells4-8. These data suggest that regions with cleaved hydrogen bonds appear in DNA during replication. Recently, it has been demonstrated that N-cyclohexyl-N-' (4-methylmorpholinium)ethylcarbodiimide (CME-carbodiimide) selectively reacts with denatured DNA, adding at its thymine and. guanineg,l°; the addition hinders the hydrolysis of modified DNA with pancreatic deoxyribonuclease (EC 3.1.4.5) and snake venom phosphodiesterase (EC 3.1.4.1). Treatment of partially denatured DNA with CMEAbbreviation: CME-, N-cyclohexyl-N'-(4-methylmorpholinium)ethyl. Biochim. Biophys. Acta, 149 (1967) 603-606