Mutual displacement in protein chromatography 



HANS G. BOMAN 



Institute of Biochemistry, University of Uppsala 



Similar substances have sometimes a marked tendency to compete for the 

 same sites on an adsorbent. This mechanism was in 1943 used for chromato- 

 graphic separations by Tiselius^ who showed that when a mixture of sucrose 

 and maltose were eluted with 0-5% phenol, the sugars came out from the 

 column as narrow zones, bordering each other. This type of chromatography 

 was named displacement development. 



Ten years later. Polis and Shmukler^ were the first to describe and utilize 

 protein-protein displacement in their purification of coloured milk proteins 

 with calcium phosphate columns. Unfortunately, they did not give any figures 

 illustrating their displacement experiments and nobody else seems to have 

 made use of their interesting findings. 



Less than two years ago, when working out the technique for protein 

 chromatography on the anion exchange resin Dowex 2,^ we found that 

 when a mixture of horse-radish enzymes was eluted with a concentrated 

 buffer, peroxidase and acid phosphatase were separated into two narrow 

 bands as shown in Fig. 1, upper part. The same separation has later been 

 obtained also on a cellulose anion exchanger. Fig. 1, lower part.* 



In ideal displacement, the length of the zone should be proportional to 

 the amount of material, and the concentration should depend on the affinity 

 constant of the substance and the concentration of the displacing agent. ^ 

 When the loading of the columns were varied, we obtained variations in 

 peak width indicating that protein-protein displacement was a major factor 

 contributing to the separation of peroxidase and the first phosphatase. This 

 was different with the second phosphatase which obviously was eluted by 

 the chloride concentration. 



However, formally, these experiments (Fig. 1) could be characterized as 

 elution with a very steep gradient of chloride and, in fact, the referee of our 

 second paper* did never believe that any protein-protein displacement was 

 demonstrated by our experiments. 



It was therefore thought to be of interest to show that a moderately ad- 

 sorbed enzyme, prostatic acid phosphatase, could be desorbed by a more 

 strongly adsorbed protein. Dowex 2 was chosen as adsorbent only because 

 of the fact that we had elution data for a variety of proteins just on this 

 resin. 3 The dimensions of the column were 19x1-2 cm. The buffer con- 



