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Desalting of mono- and oligonucleotides
SHORT COMMUNICATIONS
217
Desalting of mono- and oligonucleotides The isolation and characterization of mono- and oligonucleotides often necessitate the use of inorganic salts which m a y be later removed using charcoal I or ion-exchange resins 2. As an alternative, the nucleotide material m a y be desalted with DEAE-cellulose. A solution of 20 ml of 0.5 M ammonium sulfate containing an A2e0 m~ = 200 each of the mononucleotides of adenine, guanine, cytidine, and uracil (Nutritional Biochemicals Corp., Cleveland, Ohio) was diluted I : Io with o.or M ammonium carbonate (pH 8.6). At a flow rate of I8O ml/h, this diluted solution was then passed through a 4 × I7 cm (internal-diameter × height) column which had been packed with 20 g of DEAE-cellulose s* at a pressure of 15 lbs/in 2 and pre-equilibrated with o.oi M ammonium carbonate (pH 8.6). After the sample had passed into the column, the elution was continued with the same ammonium carbonate buffer until the ammonium sulfate 'was removed, i.e., until the conductivity of the influent and effluent buffer was the same. The mononucleotides were then quantitatively recovered b y elution with o. 7 M ammonium carbonate (pH 8.6). After lyophilization the residue was dissolved in water; conductivity measurements established the absence of ammonium sulfate. NaC1 could replace (NH4)2SO l at the same concentrations. When 20 ml of 0.5 M sodium phosphate (pH 7.0) were "desalted", only 7 ° % of the total A2s 0 was eluted with 0.7 M ammonium carbonate. However, the recovery of the mononucleotides was raised to 98 % when o.I M, rather than 0.5 M sodium phosphate was employed. In other experiments, tetranucleotides such as CpUpUpGp and ApUpUpGp 4 were quantitatively recovered from 20 ml of 2 M ammonium sulfate and o.I M sodium phosphate (pH 7.0) b y the above procedure. This desalting method should be useful for the removal of NaC1 after ion-exchange chromatography 5 of oligonucleotides which are not retained b y dialysis membranes, and difficult to elute quantitatively from charcoal. Similarly, such oligonucleotides m a y be desalted after paper chromatography with ammonium sulfate s, 7. As an alternative, mixtures of compounds, such as complete pancreatic ribonuclease digests of RNA m a y also be quantitatively recovered in a salt-free form. In this case, large oligonucleotides ("core" material) are eluted b y 2.o M instead of o. 7 M ammoniium carbonate (pH 8.6).
Laborat~rty o/ Biochemistry, National Cancer Institute, National Institutes o/ Health, Bethesda Md. (U.S.A.) x 2 8 4 6 6
G . W . RUSHIZKY H . A . SOBER
R. K. CRANE AND F. LIPMANN J. Biol. Chem., 2Ol (1953) 235. W. E. COHN AND F. J. BOLLUM, Biochim. Biophys. Acla, 48 (1961) 588. E. A. PETERSO~r AND H. A. SOBER, J. Am Chem. Sot., 78 (1956) 751. G. W. RUSHIZKY AND H. A. SOBER, J. Biol. Chem., in t h e press. E. VOLKIU AND W. E. COHN, J. Biol. Chem., 205 (1953) 767 • a . MARKHAM AND J. D. SMITH, Biochem. J., 52 (I952) 558. G. W. R u s m z K Y AND C. A. KNIGHT, Virology, I I (196o) 236.
Received August 2Ist, 1961 * B r o w n Co., Berlin, N. H., Selectacel L o t No. 1143, 0.88 rnequiv. N/g. Before use, fines .were r e m o v e d b y repeated d e c a n t a t i o n a n d the exchanger was w a s h e d as described.
Biochim. Biophys. Acta, 55 (I962) 2I 7
217
Desalting of mono- and oligonucleotides The isolation and characterization of mono- and oligonucleotides often necessitate the use of inorganic salts which m a y be later removed using charcoal I or ion-exchange resins 2. As an alternative, the nucleotide material m a y be desalted with DEAE-cellulose. A solution of 20 ml of 0.5 M ammonium sulfate containing an A2e0 m~ = 200 each of the mononucleotides of adenine, guanine, cytidine, and uracil (Nutritional Biochemicals Corp., Cleveland, Ohio) was diluted I : Io with o.or M ammonium carbonate (pH 8.6). At a flow rate of I8O ml/h, this diluted solution was then passed through a 4 × I7 cm (internal-diameter × height) column which had been packed with 20 g of DEAE-cellulose s* at a pressure of 15 lbs/in 2 and pre-equilibrated with o.oi M ammonium carbonate (pH 8.6). After the sample had passed into the column, the elution was continued with the same ammonium carbonate buffer until the ammonium sulfate 'was removed, i.e., until the conductivity of the influent and effluent buffer was the same. The mononucleotides were then quantitatively recovered b y elution with o. 7 M ammonium carbonate (pH 8.6). After lyophilization the residue was dissolved in water; conductivity measurements established the absence of ammonium sulfate. NaC1 could replace (NH4)2SO l at the same concentrations. When 20 ml of 0.5 M sodium phosphate (pH 7.0) were "desalted", only 7 ° % of the total A2s 0 was eluted with 0.7 M ammonium carbonate. However, the recovery of the mononucleotides was raised to 98 % when o.I M, rather than 0.5 M sodium phosphate was employed. In other experiments, tetranucleotides such as CpUpUpGp and ApUpUpGp 4 were quantitatively recovered from 20 ml of 2 M ammonium sulfate and o.I M sodium phosphate (pH 7.0) b y the above procedure. This desalting method should be useful for the removal of NaC1 after ion-exchange chromatography 5 of oligonucleotides which are not retained b y dialysis membranes, and difficult to elute quantitatively from charcoal. Similarly, such oligonucleotides m a y be desalted after paper chromatography with ammonium sulfate s, 7. As an alternative, mixtures of compounds, such as complete pancreatic ribonuclease digests of RNA m a y also be quantitatively recovered in a salt-free form. In this case, large oligonucleotides ("core" material) are eluted b y 2.o M instead of o. 7 M ammoniium carbonate (pH 8.6).
Laborat~rty o/ Biochemistry, National Cancer Institute, National Institutes o/ Health, Bethesda Md. (U.S.A.) x 2 8 4 6 6
G . W . RUSHIZKY H . A . SOBER
R. K. CRANE AND F. LIPMANN J. Biol. Chem., 2Ol (1953) 235. W. E. COHN AND F. J. BOLLUM, Biochim. Biophys. Acla, 48 (1961) 588. E. A. PETERSO~r AND H. A. SOBER, J. Am Chem. Sot., 78 (1956) 751. G. W. RUSHIZKY AND H. A. SOBER, J. Biol. Chem., in t h e press. E. VOLKIU AND W. E. COHN, J. Biol. Chem., 205 (1953) 767 • a . MARKHAM AND J. D. SMITH, Biochem. J., 52 (I952) 558. G. W. R u s m z K Y AND C. A. KNIGHT, Virology, I I (196o) 236.
Received August 2Ist, 1961 * B r o w n Co., Berlin, N. H., Selectacel L o t No. 1143, 0.88 rnequiv. N/g. Before use, fines .were r e m o v e d b y repeated d e c a n t a t i o n a n d the exchanger was w a s h e d as described.
Biochim. Biophys. Acta, 55 (I962) 2I 7