Question

When performing his experiments on protein refolding, Christian Anfinsen obtained a quite different result when reduced ribonuclease was reoxidized white it was still in 8 M urea and preparation was then dialyzed to remove the urea. Ribonuclease reoxidized this way had only 1% of the enzymatic activity of the native protein. Why were the outcomes so different when reduced ribonuclease was reoxidized in the presence and absense of urea?

Answer 1

Ribonuclease (RNase) is an enzyme that is held in its tertiary and quaternary form by the presence of disulphide linkages. Unlike other proteins, RNase does not degade by simply heating to temperatures of nearly 100oC. For the degradation of RNase, treatment with denaturing agents that break the disulphde linkages such as urea or mercaptoethanol, followed by heating to 100oC is required.

Christian Anifsen is performing the reoxidation of the reduced ribonuclease, while it was still dissolved in urea. This means the urea is still denaturing the protein during the reoxidation. When the urea is removed by dialysis, the denaturation stops and protein is left with a little activity, which in this case is 1%.

In order to retain nearly the full activity of Ribonuclease, Christian has to performing the reoxidation after the removal of the urea by dialysis.