Simplified diethylaminoethylcellulose chromatography of ribonucleosides

286

SHORT

Simplified

COMMUNICATIOh-S

Diethylaminoethylcellulose of

Chromatography

Ribonucleosides

A rapid, simple procedure for the separation and quantitation of rihonucleosides was recently described by van den Bos, van Kamp, and Planta (1). They first separated nucleosides from nucleotides on a DEAEcellulose column (Serva, Heidelberg, Germany) and then separated the nucleosides from each other on a Bio-Gel P-2 column (Bio-Rad, Richmond, Calif. 1. When we followed this procedure with a mixture of known ribosides using a Whatman DE-52 column (Reeve Angel, Clifton, New
ADENOSINE n CYTIDINE ,

40 -

GUANOSINE

XT 60 -

URIDINE

80 100

1 50

0

+150

100

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MINUTES

FIG. 1. Separation of ribonucleosides on Whatman DE-52. A riboside solution prepared from ToruZu ribonucleic acid (Calbiochem, Los Angeles, Calif.) by alkaline hydrolysis followed by treatment with Escherichia co2i alkaline phosphatase was placed on a freshly packed 0.9 X 9 cm DE-52 column. The column was previously equilibrated with 1.5 M ammonium hydroxide. The ribosides were eluted at room temperature with 1.5M ammonium hydroxide at a flow rate of 1 ml/min. The effluent was monitored at 260 nm with a flow cell in a Turner 330 spectrophotometer and a Heath recorder. Ribotides can be removed from the column with 0.3 n/r ammonium bicnrhonnte. pH 10.6.

REFERENCE 1. VAN

DEN

( 1970) .

Ros,

R. C.,

v.4~

E~A?MP,

G. J.,

AND

PLAPI’TA,

R. J.. Ann/.

Biochem.

35, 32

SHORT

CONMI-KICATIOKS

JOSEPH

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BARBARA

SMITH

Precipitates Carrier: Biological

Components

During immunochemical work on antigens in human normal and malignant cells, a separation procedure was developed which in one step resulted in a 20-fold increase of specific activity. This procedure appears to be applicable also in other situa.tions in which purification of delicate biological substances is attempted. In principle, the procedure employs the following steps: -4 mixture of proteins or conjugated proteins is precipitated by an isoelectric adjustment of pH. The precipitate is mixed with a suitable gel (for review, see ref. 2’) such as Sephadex G-25 (Pharmacia) or Bio-Gel P-2 (Bio-Rad). This mixture is layered on top of a column containing an identical gel of the same isoelectric pH. The column is then subjected to pH gradient clution under control of flow rate and temperature. The separation of molecular speciesthat takes place can be visualized in the following way: The precipitated proteins are exposed to a flow of ions which brings one protein after another in solution. The time required for solubilization of the various proteins will depend on the ionic strength, pH, temperature, and the flow rate. Since the distribution coefficient of each dissolved protein is lower than that of the ionic gradient, the protein will travel faster in the gel than will the gradient (2). In consequence, the protein will reprecipitate and become stationary until redissolved by the approaching ionic front. This process repeat’s itself an