Waldo E. Cohn

Rapid ion-exchange chromatographic microanalysis of ultraviolet-absorbing materials and its application to nucleosides☆

The major nucleosides obtained by hydrolysis of RNA are quantitatively assayed in less than 1 hr at the level of 1 or more nmoles by cation-exchange chromatography and a sensitive, continuous ultraviolet-absorbance monitoring device. A uniform cation-exchanger bead preparation (17.5 ± 2 μm diameter) permits operation at low (6–16 psi) pressures and rapid flow rates (1 ml/cm2 min) without loss of resolution. Analytical sensitivity is enhanced by improved flow cell and electronic circuit designs. A multi wavelength monitoring unit incorporated into the monitoring system aids in assaying substances of differing ultraviolet-absorption characteristics. Minor or altered nucleosides (20) and purine and pyrimidine bases (8) are separable, with few exceptions, by means of the single solvent chosen for the elution. The parameters determining the effectiveness of the separations have been explored and are expressed in terms permitting design modifications to improve the separation of refractory pairs or shorten the analysis time or both.

Analytical separation of nucleosides by anion-exchange chromatography: Influence of pH, solvents, temperature, concentration, and flow rate☆

The anion-exchange separation of nucleosides is influenced by pH, alcohols, temperature, ionic strength, and flow rate. The influence of each of these conditions, leading to choice of optimal operating conditions, is explored. The results are expressed quantitatively in terms of resolution and theoretical plate height. These basic parameters are derived and their use in assessing separation procedures is demonstrated.

Acid degradation products of deoxyribonucleic acid.

Abstract The acid hydrolysis of calf thymus DNA by normal HCl at 100° results primarily in nucleotides containing one more phosphate than pyrimidine, a number of which have been isolated and identified by ion-exchange chromatography. This is consistent with an elimination mechanism that leaves on the pyrimidine residue those phosphates formerly lying between purine and pyrimidine residues. It also indicates that the loss of purine residues is a necessary prerequisite to the rupture of the polynucleotide chain by mild acidic hydrolysis. The presence of nucleotides containing equal amounts of pyrimidine and phosphate may be caused by the hydrolysis of phosphate from the initial products. About 33% of the phosphate appears as inorganic phosphate after 1 hour of hydrolysis; the bulk of this appears to be phosphate originally lying between purine residues in the original DNA. There is a slow rise to a new plateau of 50% at 3 to 4 hours. The sum of inorganic plus monoesterified phosphate remains constant from 1 hour to 4 hours, indicating a slow hydrolysis of monoester phosphate, presumably from the 3′ positions. The presence of all varieties of mono-, di-, and trinucleotide sequences that are possible from cytidylic and thymidylic acids indicates that all possible sequences of pyrimidine and of purine nucleotides exist in thymus DNA.

THE SEPARATION OF BIOCHEMICALLY IMPORTANT SUBSTANCES BY ION EXCHANGE CHROMATOGRAPHY

Although ion exchange, as a chemical reaction, is over a century old and ion-exchange reactions (e.g., water softening) have been commonplace in chemical technology for decades, it has been only a few years since the first demonstration of ion-exchange chromatography of a precision capable of separating the closely related members of such families as the rare earths and the amino acids. With these demonstrations, which could not have taken place before the invention of the modern, stable, reproducible, synthetic exchangers, ion-exchange chromatography has emerged as a powerful analytical and preparative tool with which to approach those biochemical separation problems which had yielded only partially or not at all to classical methods. I t is in the separation of each of the individual members of the conventional biochemical mixtures that ion exchange has made and promises to make its greatest contributions to biochemistry, contributions which far exceed in general utility the older applications to the isolation of single substances or of groups of related substances. Other papers in this monograph deal with basic principles and nonbiochemical or nonchromatographic applications of ion exchange. This paper is limited to an exposition of the more recently reported chromatographic ion exchange separations of the members of families of biochemical importance, specifically, amino acids, purine and pyrimidine bases, nucleotides, sugars and sugar derivatives, carboxylic acids, and proteins. No attempt at comprehensive historical development or bibliographic citation is made. For this the reader is referred to the report by Cannan’ before this Academy in 1946 and to the review by Block2 in 1949, both concerning the ion exchange properties of amino acids; to those of Applezweig3 and of Applezweig and Nachod4 dealing with general biochemical and physiological applications and separations; and to the more recent comprehensive reviews of Kunin5 and of Kressman.6

Introductory Remarks by Chairman

This particular symposium on tracer methodology coincides almost exactly with the twentieth anniversary of the first shipment of radioactive material by the Oak Ridge National Laboratory to the general scientific public. It might be appropiate, therefore, to pause for a glance back to the years when the radioisotope production facilities at Oak Ridge, which underlie so much of biological research in the past two decades, were being developed [1]. Since my concern with radioisotopes, no more than average since then, played a central role in that development, such a glance may give your chairman a bit of “status” in this company [2].