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1. Introduction to Biochemistry4h 34m
2. Water3h 23m
3. Amino Acids8h 10m
4. Protein Structure10h 4m
5. Protein Techniques14h 5m
6. Enzymes and Enzyme Kinetics13h 38m
7. Enzyme Inhibition and Regulation 8h 42m
8. Protein Function 9h 41m
9. Carbohydrates7h 49m
10. Lipids5h 49m
11. Biological Membranes and Transport 6h 37m
12. Biosignaling9h 45m
Review 1: Nucleic Acids, Lipids, & Membranes2h 47m
Review 2: Biosignaling, Glycolysis, Gluconeogenesis, & PP-Pathway3h 12m
Review 3: Pyruvate & Fatty Acid Oxidation, Citric Acid Cycle, & Glycogen Metabolism2h 26m
Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation1h 48m

5. Protein Techniques

Edman Degradation

5. Protein Techniques

Edman Degradation: Videos & Practice Problems

Video Lessons Practice Worksheet

Topic summary

The Edman degradation procedure is a protein sequencing technique that identifies the N-terminal amino acid of a peptide chain through a cycle of three chemical reactions. Initially, the peptide is treated with phenyl isothiocyanate (PITC) under basic conditions, followed by trifluoroacetic acid, which releases the N-terminal amino acid as a derivative. This derivative is then treated with aqueous acid to form a stable PTH amino acid, which can be analyzed using HPLC. This process is repeated until the entire peptide sequence is determined, emphasizing the importance of peptide purification and the sequential nature of the reactions.

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Edman Degradation

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Edman Degradation Video Summary

Edmond degradation is a vital protein sequencing technique developed by Per Edmond in the 1960s, specifically designed to analyze one polypeptide chain at a time. Before applying this method, it is essential to isolate a small peptide of interest using protein purification techniques. The process is limited to small peptides due to the efficiency of the reactions involved, which will be discussed in further detail later.

The Edmond degradation procedure consists of a cycle of three distinct chemical reactions that sequentially remove and identify the N-terminal amino acid residue from the peptide. This method is often referred to as N-terminal peptide sequencing because it starts at the N-terminal end and progresses toward the C-terminal end of the peptide chain.

The three reagents used in this process are:

Phenyl isothiocyanate (PITC): This is the first reagent, which reacts under basic conditions (approximately pH 9) to label the N-terminal amino acid.
Trifluoroacetic acid (CF₃COOH): The second reagent that facilitates the release of the N-terminal amino acid residue from the peptide chain.
Aqueous acid (H₃O⁺): The third reagent that converts the released amino acid into a more stable derivative known as PTH (phenylthiohydantoin) amino acid.

For example, consider a decapeptide (a peptide with 10 amino acid residues) where the sequence is unknown. The process begins with treatment using PITC, followed by trifluoroacetic acid, which results in the release of the N-terminal amino acid as a PTH derivative. This derivative can then be identified using techniques such as high-performance liquid chromatography (HPLC). If the first identified residue is alanine, the process continues by returning the remaining peptide (now with one less amino acid) back to the initial step to identify the next amino acid in the sequence.

Each cycle of Edmond degradation reveals one amino acid residue, and the entire process is repeated until the complete sequence of the peptide is determined. The final product analyzed for identification is the PTH amino acid, which is crucial for confirming the identity of the N-terminal residue. This systematic approach allows for the comprehensive sequencing of peptides, making Edmond degradation a fundamental technique in biochemistry and molecular biology.

Problem

The peptide Leu—Cys—Arg—Ser—Gln is subject to Edman degradation. Products of the 1^st cycle include:

PTH—Leu, PTH—Cys, PTH—Arg, PTH—Ser, and PTH—Gln

PTH—Leu—Cys—Arg—Ser—Gln

PTH—Gln and Leu—Cys—Arg—Ser

PTH—Leu and Cys—Arg—Ser—Gln

Problem

Suppose you isolated a nonapeptide (9 amino acid residues) from a patient's blood. Reaction of the nonapeptide with FDNB followed by acid hydrolysis produces a DNP-product with a sulfhydryl R-group, indicating that:

C-terminal residue of the nonapeptide is Cysteine.

N-terminal residue of the nonapeptide is Cysteine.

N-terminal residue of the nonapeptide is Tyrosine.

C-terminal residue of the nonapeptide is Threonine.

C-terminal residue of the nonapeptide is Tyrosine or Glycine.

Problem

Treatment of the nonapeptide from the previous problem (1-1) with CNBr produces a tetrapeptide containing the N-terminal amino acid and a pentapeptide. After one round of Edman degradation on the pentapeptide, a product is produced that contained a nonpolar, aliphatic R group, meaning that the pentapeptide has:

N-terminal I.

N-terminal S.

C-terminal E.

N-terminal H.

N-terminal M.

Problem

The second and third rounds of Edman degradation on the same pentapeptide from the problem above (1-2) produced products with aliphatic alcohol groups, meaning that the pentapeptide had:

S and H.

I and Y.

M and C.

T and S.

M and Y.

Problem

Hydrazinolysis of the same pentapeptide from the problems above produced modified amino acids & a free α-amino acid with an aromatic-alcohol group. Combining the info from these problems, the pentapeptide is most likely:

STIRY.

HTSMY.

TISMY.

ISTRY.

ISMRY.

Problem

Deduce the entire sequence of the original nonapeptide using the following hint and the information from the previous four practice problems (1-1 through 1-4). Hint: The sequence of the nonapeptide using the one-letter amino acid codes reveals a relevant academic subject.

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What is Edman degradation and how does it work?

Edman degradation is a protein sequencing technique that identifies the N-terminal amino acid of a peptide chain. The process involves a cycle of three chemical reactions. First, the peptide is treated with phenyl isothiocyanate (PITC) under basic conditions, which reacts with the N-terminal amino acid. Next, trifluoroacetic acid is used to cleave the N-terminal amino acid as a derivative. Finally, this derivative is treated with aqueous acid to form a stable PTH amino acid, which can be analyzed using high-performance liquid chromatography (HPLC). This cycle is repeated to determine the entire peptide sequence.

Why is Edman degradation limited to small peptides?

Edman degradation is limited to small peptides because the efficiency of the reaction decreases with longer peptide chains. Each cycle of the degradation process removes one N-terminal amino acid, and the accuracy of identifying each amino acid diminishes as the number of cycles increases. This is due to incomplete reactions and side reactions that can occur, leading to a loss of sequence information. Therefore, peptides typically need to be relatively small, usually less than 50 amino acids, to ensure accurate sequencing.

What are the reagents used in Edman degradation?

Edman degradation uses three main reagents in its cycle of reactions. The first reagent is phenyl isothiocyanate (PITC), also known as the Edman reagent, which reacts with the N-terminal amino acid under basic conditions. The second reagent is trifluoroacetic acid (CF₃COOH), which cleaves the N-terminal amino acid as a derivative. The third reagent is aqueous acid (H₃O⁺), which converts the derivative into a stable PTH amino acid for identification using high-performance liquid chromatography (HPLC).

How is the N-terminal amino acid identified in Edman degradation?

In Edman degradation, the N-terminal amino acid is identified through a series of chemical reactions. Initially, the peptide is treated with phenyl isothiocyanate (PITC) to form a derivative of the N-terminal amino acid. This derivative is then cleaved from the peptide using trifluoroacetic acid. The released derivative is treated with aqueous acid to form a stable PTH amino acid. This PTH amino acid is then analyzed using high-performance liquid chromatography (HPLC) to determine its identity. This process is repeated for each subsequent N-terminal amino acid in the peptide sequence.

What is the role of high-performance liquid chromatography (HPLC) in Edman degradation?

High-performance liquid chromatography (HPLC) plays a crucial role in Edman degradation by identifying the PTH amino acid formed after each cycle of the degradation process. Once the N-terminal amino acid is cleaved and converted into a stable PTH derivative, HPLC is used to separate and analyze this derivative. The retention time and specific characteristics of the PTH amino acid in the HPLC system allow for its identification. This information is used to determine the sequence of the peptide, one amino acid at a time.

Previous Topic: Peptidases Next Topic: Edman Degradation Sequenator and Sequencing Data Analysis

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Edman Degradation: Videos & Practice Problems

Edman Degradation

Edman Degradation Video Summary

The peptide Leu—Cys—Arg—Ser—Gln is subject to Edman degradation. Products of the 1st cycle include:

Suppose you isolated a nonapeptide (9 amino acid residues) from a patient's blood. Reaction of the nonapeptide with FDNB followed by acid hydrolysis produces a DNP-product with a sulfhydryl R-group, indicating that:

The second and third rounds of Edman degradation on the same pentapeptide from the problem above (1-2) produced products with aliphatic alcohol groups, meaning that the pentapeptide had:

Hydrazinolysis of the same pentapeptide from the problems above produced modified amino acids & a free α-amino acid with an aromatic-alcohol group. Combining the info from these problems, the pentapeptide is most likely:

Deduce the entire sequence of the original nonapeptide using the following hint and the information from the previous four practice problems (1-1 through 1-4). Hint: The sequence of the nonapeptide using the one-letter amino acid codes reveals a relevant academic subject.

Do you want more practice?

Here’s what students ask on this topic:

What is Edman degradation and how does it work?

Why is Edman degradation limited to small peptides?

What are the reagents used in Edman degradation?

How is the N-terminal amino acid identified in Edman degradation?

What is the role of high-performance liquid chromatography (HPLC) in Edman degradation?

Your Biochemistry tutor

The peptide Leu—Cys—Arg—Ser—Gln is subject to Edman degradation. Products of the 1^st cycle include: