Question

1)

• The isoelectric pH of RuBISCO is around 6. The pH of the elution buffer for this experiment is 8. What will be the elution profile of the chromatography if you use a highly acidic buffer (pH=3)?

2)• In this experiment we used an anion exchanger. What will be the elution profile of the chromatography if we had used a cation exchanger?

Answer 1

structure of a protein is dependent of the pH because the chains that conform it can have different groups, these can be charged negatively or positively. The sum of all these charges is the net sum and it has an effect on the protein, in higher pH the protein is has a negative net sum leading to aggregation. On the other hand, on lower pH than the isoelectric point the protein will have a positive sum and this will lead to segregation and it can even unfold the protein, causing it to lose the tertiary structure. If this happens there is a chance for hydrophilic groups to aggregate generating a much bigger structure.

In this experiment using a buffer of pH=3 will change the profile by making the low and high molecular weights peaks less pronounced and increasing the eluting time. Because some of the protein will aggregate when losing the structure but some of it will not aggregate and most of will reaming somewhere in between, the resulting profile will be closer to an horizontal line with the peaks more to the right side than the profile with buffer pH=8.

The use of an anion exchanger means Rubisco is positively charged in pH=8 so the molecule can be attracted to the Colum. If you instead use a cation exchanger in pH=8 then the column will not have affinity for the Rubisco but if you use the buffer pH=3 then you will have affinity, because the molecule will become negatively charged.