Edman degradation | Edman Sequencing

Summary

The script introduces Edman degradation, a technique for protein sequencing that identifies amino acids in a peptide chain. It involves labeling and cleaving the N-terminal amino acid using phenyl isothiocyanate, Edman's reagent, under mild alkaline conditions. The resulting derivative is extracted and acidified to form a stable PTH amino acid derivative, identifiable by HPLC. This method is iterative, allowing for the sequence determination of up to 30 amino acids from the peptide's end.

Takeaways

• 🔬 Edman degradation is a method used for sequencing amino acids in proteins.
• 🔗 The process involves labeling and cleaving the N-terminal amino acid of the peptide.
• 🧪 The labeled amino acid is identified through techniques like chromatography or electrophoresis.
• 💡 Phenylisothiocyanate, also known as Edman’s reagent, is used for labeling the N-terminal amino acid.
• 🧼 The reaction between Edman’s reagent and the N-terminal amino acid occurs under mild alkaline conditions.
• 🔥 Under heat and specific conditions, a phenylthiohydantoin (PTH) derivative is formed.
• 🌡️ The PTH derivative is cleaved from the peptide and extracted using organic solvents.
• 🧊 The extracted derivative is acidified to form a stable PTH-amino acid.
• 🔍 The PTH-amino acid can be identified using chromatography techniques like HPLC.
• 🔢 Edman degradation can sequence up to 30 amino acids from the N-terminal of the peptide.

Q & A

What is Edman degradation?

Edman degradation is a method used for the sequencing of amino acids in a protein. It involves the stepwise identification of amino acids from the N-terminus of the peptide.

How does the Edman degradation process begin?

The process begins by labeling the N-terminal amino acid of the peptide with a reagent, which is then cleaved from the peptide.

What is the reagent used for labeling the N-terminal amino acid in Edman degradation?

The reagent used is phenyl isothiocyanate, also known as Edman's reagent.

What conditions are required for the reaction between Edman's reagent and the N-terminal amino acid?

The reaction occurs under mild alkaline conditions.

What is produced as a result of the reaction between Edman's reagent and the N-terminal amino acid?

The reaction produces a phenylthiocarbamoyl (PTC) derivative of the N-terminal amino acid.

How is the PTC derivative cleaved from the parent peptide?

The PTC derivative is cleaved under heat and acidic conditions and then extracted by organic solvents.

What happens to the PTC derivative after it is extracted?

After extraction, the PTC derivative is acidified to form a stable PTH (phenylthiohydantoin) amino acid derivative.

How is the PTH amino acid derivative identified?

The PTH amino acid derivative can be identified using chromatography techniques such as High-Performance Liquid Chromatography (HPLC).

What is the maximum number of amino acids that can be identified using the Edman degradation method?

The maximum number of amino acids that can be identified by this technique is generally under 30.

How can the sequence of the peptide be determined using Edman degradation?

The sequence of the peptide can be determined by repeating the Edman degradation process, progressively identifying each amino acid from the N-terminus.

What are some of the limitations of the Edman degradation method?

While Edman degradation is a powerful tool, its limitations include the difficulty in identifying more than 30 amino acids and the potential for incomplete reactions or side reactions.