Question

Why can the Edman degradation not be used effectively with very long peptides? Hint: Think about the stoichiometry of the peptides and the Edman reagent and the percent yield of the organic reactions involving them.

Short answer

Edman degradation is ineffective for long peptides due to decreasing yield and accumulated errors over many cycles.

Step-by-step solution

Understand Edman Degradation

Edman degradation is a method used for sequencing amino acids in a peptide. The Edman reagent, phenyl isothiocyanate, reacts with the amino-terminal residue of the peptide, cleaving it for identification.

Consider Stoichiometry

For each cycle of Edman degradation, a single amino acid is removed from the peptide and identified. Each cycle must be perfectly stoichiometric for accurate sequencing.

Analyze Percent Yield

The reaction yield of each cycle is not 100%. Typically, the yield decreases with each successive cycle due to incomplete reactions, losses, and side reactions.

Assess the Cumulative Effect

For long peptides, the cumulative effect of imperfect yields in multiple cycles results in an accumulation of errors. This means the accuracy and reliability of sequencing decrease significantly as the length of the peptide increases.

Conclusion

Because of the decreasing yield and accumulated errors in multiple cycles, Edman degradation is not effective for sequencing very long peptides.

Key concepts

Amino Acid Sequencing

Amino acid sequencing is the process of identifying the order of amino acids in a peptide or protein. This sequence determines the protein's structure and function.

• The primary structure of a protein is a linear sequence of amino acids.
• Each amino acid is linked to another via peptide bonds.
• Methods like Edman degradation help in determining these sequences.

In Edman degradation, amino acids are sequentially removed from the peptide. This is useful for figuring out the protein's primary structure.
Understanding amino acid sequences is critical in fields like biochemistry, molecular biology, and medicine.

Phenyl Isothiocyanate

Phenyl isothiocyanate (PITC) is a chemical reagent crucial for the Edman degradation method.

• PITC reacts with the amino-terminal residue of a peptide.
• When PITC binds, it forms a phenylthiocarbamoyl derivative.
• This reaction cleaves the terminal amino acid which can then be identified.

The specific reaction with PITC allows the sequential identification of amino acids from the N-terminal end. This makes it easier to determine the sequence one amino acid at a time. In summary, PITC is essential for breaking down and identifying parts of the peptide chain.

Stoichiometry

Stoichiometry in the context of Edman degradation is all about the exact ratios of reactants and products.

• Each cycle needs one molecule of phenyl isothiocyanate for each amino acid.
• If the reaction stops being perfectly stoichiometric, the sequencing can become inaccurate.

In an ideal situation, every single amino acid should react and be identified in each step.
Any deviation can lead to errors, making the sequence harder to read correctly.
Stoichiometry, therefore, ensures that the reactions go as planned for accurate results. The importance here is maintaining the perfect balance of reactants to ensure precise sequencing steps.

Percent Yield

Percent yield refers to the efficiency of a chemical reaction.
In Edman degradation, it is rare for each cycle to have 100% yield.

• Typical yields are slightly less than 100% due to incomplete reactions and side reactions.
• With each successive cycle, the accuracy slightly drops.

As the peptide length increases, the cumulative effect of these less-than-perfect yields becomes significant. This means that sequencing errors accumulate.
Percent yield plays a huge role in dictating the reliability of longer peptide sequencing.

Peptide Length

Peptide length refers to the number of amino acids in a peptide.
In Edman degradation, short peptides (usually fewer than 50 amino acids) are easier to sequence accurately.

• Shorter peptides mean fewer cycles, reducing error accumulation.
• Longer peptides make it hard to maintain high accuracy due to decreased yield and cumulative errors as explained earlier.

Peptides over a certain length become problematic for Edman degradation.
This method becomes less effective with increasing peptide length due to these accumulated inaccuracies.