Question

In the 1950s, Christian Anfinsen demonstrated the renaturation of the protein ribonuclease (RNase) in vitro. After reduction (to reduce the disulfide links) and the addition of urea (to denature the protein), the protein was in an unfolded state. After removing the urea and the reducing agent, the protein refolded, with greater than 90% activity. If the urea were removed after oxidation occurred, the protein had less than 5% activity. Why would the protein not refold correctly if the urea were removed after the reducing agent was removed? (In other words, what would happen if the urea were removed after oxidation?)

Answer 1

Answer:

Generally disulphide bonds are  type  of covalent bonds  formed between cysteine molecules, which can  be removed by reducing agents. They are formed when weak interactive bonds are well positioned  between cysteine molecules to from disulphide bridges. The denaturation by urea   disrupts positioning of  this weak interactive bondings and therefore disulphide bridges. Thus removal of urea enabled the weak bonding interactions to correctly placed the   disulphide bonds  back between the cysteine molecules,

S-----H----H-----S       ⇒    S-------S                                    

Reduction                       Oxidation

However,  with oxidation occurring  ( i.e loss  of oxidizing agents)before removal of urea,  the –SH residues   positioning would be disrupted, affecting  the formation of disulphide bonds, and eventual   denaturation of protein.

Explanation: