Edman degradation | Edman Sequencing
Summary
TLDRThe 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.
Outlines
🧪 Edman Degradation Method for Protein Sequencing
The Edman degradation method is a technique for determining the sequence of amino acids in a protein. It involves the identification and removal of the N-terminal amino acid from a peptide chain. The process begins with the labeling of this amino acid using phenyl isothiocyanate, also known as Edman's reagent, under mild alkaline conditions. This reaction results in the formation of a phenylthiohydantoin (PTH) derivative, which is stable and can be extracted using organic solvents. Once extracted, the PTH derivative is acidified and then identified through chromatographic techniques such as high-performance liquid chromatography (HPLC). The Edman degradation can be repeated to sequence the peptide, but it is typically limited to identifying sequences of up to 30 amino acids.
Mindmap
Keywords
💡Edman degradation
💡Protein sequencing
💡N-terminal amino acid
💡Phenyl isothiocyanate (PITC)
💡Mild alkaline conditions
💡Nile blue derivative
💡Organic solvents
💡Acidification
💡PTH amino acid derivative
💡Chromatography
💡HPLC (High-Performance Liquid Chromatography)
Highlights
Edman degradation is a method for sequencing amino acids in proteins.
The method involves labeling and cleaving the N-terminal amino acid of a peptide.
Identification of the labeled amino acid is done using techniques like chromatography or electrophoresis.
Phenyl isothiocyanate is the reagent used for labeling the N-terminal amino acid.
This reagent is also known as the Edman reagent.
The reaction between Edman reagent and the N-terminal amino acid occurs under mild alkaline conditions.
The reaction produces a phenylthiohydantoin (PTH) derivative of the amino acid.
The PTH derivative is stable and can be extracted using organic solvents.
Acidification of the extracted derivative forms the PTH amino acid, which is identifiable by HPLC.
The Edman degradation process can be repeated to sequence the peptide from the N-terminal.
The maximum number of amino acids identifiable by this technique is under 30.
The method is significant for its contribution to protein sequencing.
The process is innovative in its approach to identifying amino acid sequences.
Edman degradation has practical applications in the field of biochemistry.
The technique provides a theoretical basis for understanding protein structure.
The method's impact is notable in advancing protein sequencing capabilities.
The process is unique in its ability to sequence amino acids from the N-terminal.
The technique's repeatability is a key feature for identifying long peptide sequences.
The use of organic solvents for extraction is a critical step in the Edman degradation process.
The identification of the PTH amino acid derivative is facilitated by chromatography techniques.
The mild alkaline conditions are crucial for the reaction with the Edman reagent.
The stability of the PTH derivative is essential for its identification.
Transcripts
hey guys quick marker mister basics here
let's talk about admins degradation
method for protein sequencing Edman
degradation is a method used for
sequencing of amino acids in protein in
this method the n-terminal amino acid of
the peptide is first labeled and then
cleaved from the peptide
the label amino acid is then identified
by several techniques like
chromatography or electrophoresis
the labeling of n-terminal amino acid is
done by the reagent called phenol tyo
isocyanate this is also known as Edmonds
reagent
the reaction between Edmonds reagent and
the n-terminal amino acid is done in
mild alkaline conditions
this reaction gives rise to free Nile
Taiyo garb a mild derivative
under heat and
setec conditions this derivative is
cleaved from the parent peptide and
extracted by organic solvents once
extracted it is further
acidified to form PTH amino acid
derivative which is very stable
the PTH amino acid derivative can be
easily identified by chromatography
techniques like HPLC
this reaction can be carried out several
times to find the sequence of the
peptide from the end terminal
the maximum number of amino acids that
can be identified by this technique is
under 30
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