[nj_bartel]

Question:  A denaturation/renaturation (similar to the one carried out by Anfinsen with ribonuclease) experiment was carried out using insulin.  However, in contrast to Anfinsen's results, only less than 10% of the activity of insulin was recovered when urea and BME were removed by dialysis.  (This is the level of activity you would expect if the disulfide bridges paired randomly).  In contrast, if the experiment is repeated with proinsulin, full activity is restored upon renaturation.  Explain these observations.


My idea (can't be right):  Proinsulin is a single stranded polypeptide with three intramolecular disulfide bonds.  Insulin is a double stranded polypeptide with two intermolecular disulfide bonds and one intramolecular disulfide bond.  In the case of insulin, you have numerous a and b strands in solution, and the correct a and b cysteine residues must find each other in solution before an intramolecular disulfide bond forms.  Even then, the polypeptide still has high possibility to polymerize.  Proinsulin on the other hand, has only intramolecular disulfide bonds and thus has a high probability of folding in on itself and forming those bonds before forming an intermolecular disulfide bond.

The obvious problem I see is that full activity is returned in proinsulin, but with my theory you'd still have at least SOME, probably substantial, loss.  Any words of wisdom?

[renge ishyo]

I don't know if this is the correct answer or not, but if you look at the structure of proinsulin versus the active insulin structure you will notice that there is a protective strand of amino acids covering up a disulfide bond exposed at the surface of the active insulin molecule. When proinsulin is cleaved into insulin this band is removed to expose this disulfide bond to the outside where it can be destroyed by chemical agents (the other two disulfides remain buried in both structures and are likely not involved in this matter). It could be that this is the mechanism responsible for the two different results. For an illustration, see the image on the wiki: http://en.wikipedia.org/wiki/Proinsulin#Synthesis_and_post-translational_modification

On the image you can see that in step 4 proinsulin has a protective (pink) band covering up the surface disulfide. All the disulfides are in the interior of the molecule at this point. After this band is cleaved to form active insulin shown in step 5, one of the disulfides is now exposed to the environment outside and can be chemically attacked. This would be my explanation at least 

[nj_bartel]

Thank you   Didn't realize structures being drawn like that was representative of how they were in their tertiary structure.