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.
To explain these observations, we need to understand the structure and folding of insulin and proinsulin, as well as the role of disulfide bridges in their respective folding processes.
Insulin is a hormone composed of two peptide chains, referred to as the A-chain and the B-chain, which are connected by two disulfide bridges. The correct folding and formation of these disulfide bridges is crucial for insulin to achieve its functional three-dimensional structure and maintain its activity.
In denaturation/renaturation experiments, denaturing agents like urea are used to disrupt the native structure of a protein, while reducing agents like β-mercaptoethanol (BME) break the disulfide bridges. Dialysis is then performed to remove these denaturing and reducing agents so that the protein can attempt to refold and regain its native conformation.
In the case of insulin, when denatured and then renatured without any assistance, less than 10% of its activity is recovered. This suggests that the disulfide bridges in insulin are not correctly reformed during renaturation. The random pairing of disulfide bridges results in misfolded or non-functional insulin molecules, leading to the observed low activity.
On the other hand, when proinsulin is denatured and renatured, it regains full activity. Proinsulin is the precursor of insulin and contains an additional peptide chain called the C-peptide. The folding of proinsulin involves the correct formation of three disulfide bridges, one within the A-chain and two within the B-chain, as well as the removal of the C-peptide during insulin maturation. Proinsulin has an inherent ability to correctly fold and form its native disulfide bond pattern even without assistance from additional folding catalysts or factors.
Therefore, the key difference between insulin and proinsulin in this experiment lies in their ability to correctly reform disulfide bridges during renaturation. Insulin, lacking the C-peptide and potentially other factors necessary for correct disulfide bond formation, exhibits a low recovery of activity due to random disulfide pairing. Proinsulin, with its inherent folding properties, can properly refold and regain its full activity upon renaturation.