Question

A mixture of three proteins shown below was applied to an ion-exchange column with CM-Sepharose as the stationary phase. Predict the order of elution of the proteins at neutral pH, assuming that the mixture was applied at low ionic strength and eluted with buffers of increasing ionic strength. Explain.

• Cytochrome c (12.0 kDa, pI = 10.6)
• Phosphofructokinase 1 (34.1 kDa, pI = 6.64)
• Bovine serum albumin (66.5 kDa, pI = 4.70)

Answer 1

CM-Sepharose is a weak cationic exchanger, it means that CM-Sephrose is a negetaively charged resin which binds to positively charged particles.

PI of the amino acid is a pH at which the total charge on amino acid will be Zero it means at PI amino acid is having neutral charge. if the pH is greater than PI the amno acid will be negatively charged and if pH is lower than PI the amino acid will be positively charged.

By given information

At Neutral pH. 7.00

1. Cytochrome c will have negative charge at neutral pH.

2. Phosphofructokinase will have positive charge

3. BSA(Bovine serum albumin) will be more positive than PFK .

So the order of elution will be:

Cytochrome c (first)--> Phosphofructokinase (second) --> Bovine serum albumin (last)