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?)

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: