In the 1950s, Christian Anfinsen demonstrated the renaturation of the protein ribonuclease (RNase) in vitro. After reduction and the addition of urea, the protein was in an unfolded state. After removing the urea and then the reducing agent, the protein oxidized and refolded, with greater than 90% activity. If reducing agent removal occurs before removing the urea, the protein showed less than 5% activity. Why does synthetically produced RNase refold incorrectly if the reducing agent is removed before urea removal?

The proteins primary amino acid sequence determines the path of folding to the fully functional 3-Dimensional protein. Removing the urea first, allows spontaneous and rapid correct folding of the protein, thus driving the correct disulfide bond pairing.

In contrast, removing the reducing agent first causes random disulfide bond pairing which ultimately leads to misfolded and inactive proteins.

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