274 Essays in Biochemistry- 



laboratories, including our own. In this procedure, the chromatogram 

 is begun with an eluent incapable of eluting the protein in question 

 (R f = 0) , whereupon the composition of the eluent is gradually changed 

 to one in which the protein has an R f of 1. The exact point of emer- 

 gence of the protein will thus be a function more of the rate of change 

 of the eluent than of the length of the column. Although worth-while 

 purifications can be effected in this manner, 9 ' 10 elution analysis of 

 proteins has, in our experience, been found to be superior when it can 

 be made to work properly. 



Satisfactory elution analysis has been possible with several proteins 

 on both partition and ion-exchange columns. Despite the fact that 

 these proteins are polyvalent molecules, test-tube experiments showed 

 unquestionably that, in several instances, reversible distribution be- 

 tween resin (IRC-50, in each case) and buffer existed. Moreover, the 

 magnitude of this distribution coefficient could be changed in a pre- 

 dictable fashion by altering the pH or the ionic strength of the buffer 

 phase. In short, several proteins, among them ribonuclease, 11 have 

 been found to behave much like simple substances. It is pertinent 

 to inquire why this should be so. From a logical point of view, the 

 problem really is not why most proteins chromatograph unsatisfac- 

 torily but why some behave well. Ribonuclease, for example, contains 

 a minimum of fifteen possible cationic sites (cf. Table 1) which could 

 exchange with Na+ on a buffered IRC-50 column. A clue may be 

 furnished by the electrophoretic studies of Crestfield and Allen 12 that 

 show that the isoelectric point of ribonuclease drops from pH 7.48 in 

 dilute acetate buffers to pH 5.49 in 0.2 N phosphate buffers of the type 

 used in chromatography. Apparently phosphate ions form a feebly 

 dissociable complex with ribonuclease, and it may be that the existence 

 of this complex facilitates chromatography on the ion exchange resin. 

 Complexing agents have been utilized with success in other fields, 

 notably in the case of the rare-earth elements by Speckling and his 

 associates 13 and in the case of the sugars by Khym and Zill. 14 Perhaps 

 a systematic search for agents that form complexes with proteins might 

 widen the scope of elution analysis. Whether or not chromatography 

 is the best way to procure them, it is unquestionably true that a larger 

 number of rigorously purified proteins remains one of the critical needs 

 of the protein chemist. 



In considering which proteins were likely to be good subjects for 

 detailed structural study, the fact that ribonuclease could be purified 

 chromatographically was one of the most important considerations 

 leading to its selection. Its small size (mol. wt., 14,000) and the 



