Anfinsen's Protein Folding Experiment Overview

Anfinsen's classic experiment with ribonuclease enzyme demonstrated that the information needed for a protein to fold into its correct three-dimensional structure, and thus regain its function, is contained entirely within its primary amino acid sequence.

In this experiment, ribonuclease A, which has enzymatic activity, is treated with two agents:

• $\beta$-mercaptoethanol: This reducing agent breaks the disulfide bonds (S-S bonds) within the protein by reducing them to sulfhydryl groups (-SH). Ribonuclease A has four disulfide bonds crucial for its stable structure and activity.
• 8 M urea: This is a strong denaturing agent that disrupts the protein's non-covalent interactions (like hydrogen bonds and hydrophobic interactions), causing the protein to unfold into a random coil conformation.

After this treatment, the ribonuclease is denatured and its disulfide bonds are broken. It loses its specific three-dimensional structure and, consequently, its enzymatic activity.

Regaining Ribonuclease Enzymatic Activity

The question asks which step following this denaturation will lead to regaining full enzymatic activity. Enzymatic activity requires the protein to refold into its native, active conformation with the correct disulfide bonds reformed.

Let's analyze the given options:

• A. dialyzed to remove the $\beta$-mercaptoethanol and 8 M urea: Dialysis is a method used to remove small molecules from a solution containing larger molecules (like proteins). By dialyzing the treated ribonuclease solution against a buffer without $\beta$-mercaptoethanol and urea, these denaturing agents are removed. When urea is removed, the non-covalent interactions can reform, allowing the protein chain to adopt a more structured form. When $\beta$-mercaptoethanol is removed, the environment becomes oxidizing (due to the presence of oxygen in the air), which allows the free sulfhydryl groups to reform disulfide bonds. Anfinsen found that under these conditions, the protein spontaneously refolds into its correct native structure with the correct disulfide bonds, leading to the regain of full enzymatic activity.
• B. the sample was completely oxidized in 8M urea after dialysis: If oxidation occurs in the presence of 8 M urea, the protein is still largely unfolded. While disulfide bonds might form, they would likely form randomly between incorrect pairs of cysteine residues because the protein isn't in the conformation that guides correct bond formation. This would lead to misfolded, inactive protein.
• C. trace amounts of $\beta$-mercaptoethanol was added to the dialysed sample: Adding $\beta$-mercaptoethanol maintains a reducing environment. In a reducing environment, disulfide bonds are less likely to form, or if they form, they can be quickly broken again. This would prevent the stable reformation of the necessary disulfide bonds for the active structure.
• D. 8M urea was added to the dialyzed sample: Adding 8 M urea back to the dialyzed sample would simply redenture the protein that might have started refolding. Denaturation prevents the formation of the active three-dimensional structure required for enzymatic activity.

Conclusion

According to Anfinsen's findings, removing the denaturing agents ($\beta$-mercaptoethanol and urea) by dialysis allows the denatured ribonuclease to spontaneously refold into its native conformation and reform the correct disulfide bonds in the presence of oxygen from the air. This single step leads to the regain of full enzymatic activity.

Therefore, the step that will lead to regaining full enzymatic activity of ribonuclease is dialyzing to remove the $\beta$-mercaptoethanol and 8 M urea (Option A) alone.