Question

21. A mixture of urease (pl = 5.1; MW 482,000), catalase (pl = 5.6; MW 247,500), lactoglobulin (pl = 5.2; MW 37,100) and hemoglobin (pl = 6.9; MW 64,500) were applied in a pH 6.5 buffer to a DEAE-cellulose (anion exchanger resin) chromatography column and eluted with the same buffer. What is their order of elution? How would you elute bound proteins from this type of resin? Explain your answer. (6 pts)

Answer 1

Ans: As you know that at pH=pI the net charge on the protein is zero and above pI negative charge dominates and below pI positive charge dominates. So at pH 6.5 urease has negative charge catalase has negative charge but with less intensity than urease lactoglobulin has negative charge with great intensity as compared to catalse but less intensity than urease and hemoglobin has positive charge. And as described in the question we use DEAE-cellulose as the resin which is an anion exchanger resin i.e they have net positive charge and they exchange anions based on the attractive force. So as described above out of these four proteins hemoglobin is positive charged so there will be a repulsive force between the resin and hemoglobin so it will be eluted out first, it is followed by catalase then lactoglobulin and at last urease.

To elute bound protein from this type of resin we have to use a buffer with pH less than the pI of the bound protein because at pH less than pI positive charge dominates and this will cause force of repulsion between resin and the bound protein and hence the protein will eluted out from the resin.