Question

Protein	Molecular Weight	Isoelectric point	Does the protein contain heme moiety?
1	68,000	5.5	No
2	15,000	5.0	No
3	17,500	9.5	Yes

Could gel filtration chromatography be used to separate a mixture containing proteins 1 & 3?

If gel filtration chromatography can be used, which protein would elute las? After collection fractions from the column, the absorbance of each fraction will be measured in a spectrophotometer - can both proteins 1 & 3 be monitored at 280 nm and 400 nm?

Which 2 proteins can be separated by ion exchange, but NOT gel filtration chromatography?

Which 2 proteins can be separated by gel filtration but NOT ion exchange?

Answer 1

a) Yes. It is possible to separate a mixture containing proteins 1 & 3 since it has large difference in size.

Exclusion chromatography or Gel filtration chromatography is a separation technique base on the size of the components in the mixture. The stationary phase in this chromatographic technique is porous beads of well defined size. During the process of separation the proteins can enter into these pores. Only the proteins which are small enough to enter into these pores can only stay in stationary phase. The other big sized proteins will be excluded. So in this chromatography big sized will be eluted to first (will come out of the column first) and the smallest ones last. Protein 1 will be eluted first and then protein 3 will come out of column.

Heme containing protein ( protein 3) can be monitored at 280 nm and 400 nm. Protein 1 cannot be monitored at these wavelengths.

Ion exchange chromatography

Ion exchange chromatography is a chromatographic technique to separate components based on their charge. Proteins can have many ionizable groups. These include the end amino and acid groups as well. To separate the proteins we should know its charge at pH 7. The isoelectric point of proteins the value of pH at which the protein does not have a charge. So from the pI values we will get the charge of the protein. The stationary phase using in ion exchange chromatography are charged resins. If we are using positively charged resin then during the process of separation, negatively charged proteins will bind to the stationary phase. Thus positively charged proteins will be eluted first and negatively charged last. If we are using negatively charged resin then during the process of separation, positively charged proteins will bind to the stationary phase. Thus negatively charged proteins will be eluted first and positively charged last. If pH > pI the protein has a negative charge, if pH = pI the protein has no charge and pH < pI the protein has a positive charge.

Proteins 2 & 3 cannot be separated by gel filtration chromatography because of their small size difference. But these can be separated by ion exchange chromatography. Because they have pH values which are very different.

Proteins 1 & 2 cannot be separated by ion exchange chromatography because of their small difference in pH values. But these can be separated by gel filtration chromatography because of their large in difference in size.