Chromatography of ribonuclease on carboxymethyl cellulose columns.

In the course of an investigation of the chemical phosphorylation of ribonucleasel (cf. (l)), it became necessary to develop a chromatographic procedure for the fractionation of the phosphorylated protein. The method of Hirs et al. (2), employing the weak cation exchanger Amberlite XE-64, is an extremely valuable method for the chromatographic purification of pancreatic ribonuclease, but the use of phosphate buffer, pH values below 7, and the rapid movement of the protein through the column rendered the method unsuitable for a study of acidlabile, phosphorylated ribonuclease. In the method described in this paper carboxymethyl cellulose ion exchange columns and a gradient elution technique were employed, based on the procedures of Peterson and Sober (3). Quantitative measurements were also made of the affinity of the exchanger for ribonuclease as a function of pH and ionic strength. The CM-cellulose method offers a number of advantages over the method employing the Amberlite resin. Furthermore, by this chromatographic procedure crystalline bovine pancreatic ribonuclease is fractionated into at least four enzymatically active components, in contrast to the two active components separated on the Amberlite column. After this work had been completed, a report by iqvist and Anfinsen appeared (4) describing an ion exchange chromatographic fractionation of ovine pancreatic ribonuclease on CMcellulose columns. These authors also describe an experiment in which crystalline bovine pancreatic ribonuclease was subjected to CM-cellulose chromatography with sodium phosphate as the eluting buffer between the gradient limits 0.01 to 0.1 M and pH 6:O to 7.5. Their finding of four enzymatically active components is confirmed in the present investigation carried out without the use of phosphate and avoiding acid pH values. In view of the known strong affinity of ribonuclease for multivalent anions, such as the phosphate ion (lowering of the isoelectric point of ribonuclease by 2 pH units (5)), it is of interest that the separation of the enzyme preparation by ion exchange chromatography into several components is accomplished also when a monovalent anion (such as chloride, as in the work described in this paper) is present in the buffer.