Question

You have a mixture of three proteins, Huskerase (pI=6.5), Gopherase (pI=8.6), and Badgerase (pI= 4.6), which you would like to separate by chromatographic technique(s). A native gel indicates that the three proteins have approximate molecular weights of 240 kDa, 215 kDa, and 79 kDa respectively. a) Based on this information, describe an appropriate separation strategy using chromatography. Be sure to indicate relevant conditions (pH, resin(s), etc). b) Given that Huskerase is a homotrimer, Gopherase a heterotetramer, and Badgerase a homodimer, sketch the results of a reducing SDS-PAGE experiment that you performed to examine the purity of each of your three separated proteins. (Hint: You should have a total of four lanes in your gel.) c) You suspect that Huskerase contains an intersubunit disulfide bond. Describe an experiment to test this hypothesis. Sketch out your expected results.

Answer 1

A protein is an amphoteric molecule. When its pH is more than its isoelectric point(pI) then the protein become negetively charged and move towards the positive electrode. When the pH is less than its pI the protein becomes positively charged and move towards the negetive electrode. As the protein moves through increasing pH its overall charge decreases until it reaches the pH which corresponds to its pI where the migration stops since there is no electrical attraction towards either electrode. In an anion exchanger chromatography the pH of the buffer should be greater than the pI of the proteins and in cation exchanger pH should be less than the pI of the proteins. In the above case given we can use an anion exchanger with pH of the buffer being 5-9. The anion exchanger Q(quaternary resin) and diethylaminoethane (DEAE) are the two types of anion exchanger which can be used.