Anfinsen used the protein RNase A (has a single polypeptide chain with 8 cysteines). Anfinsen wanted to know if there are substances within cells (enzymes?) that cause other proteins to fold properly into their appropriate 3D shapes.

His first step was to apply mercaptoethanol alone to the RNase and that caused the S-S bonds between the cysteins to break, but no unfolding. Then he applied urea alone and that caused unfolding but didn’t change the sequence of the amino acids. Both mercaptoethanol and urea caused the RNase to lose it’s enzymatic function.

he decided to remove these reagents (urea and mercaptoethanol) in 2 ways:
Method 1: remove mercaptoethanol first and then remove urea
Method 2: remove urea first and then remove mercaptoethanol

Method 1 resulted with very little activity in the RNase.

Method 2 resulted with the RNase regaining almost all of it’s enzymatic function.