Question

You are in the process of determining the amino acid sequence of a peptide. After trypsin digestion followed by the Edman degradation, you see the following peptide fragments: $$Leu-Gly-Arg$$ $$Gly-Ser-Phe-Tyr-Asn-His$$ $$Ser Glu-Asp-Met-Cys-Lys$$ $$Thr-Tyr-Glu-Val-Cys-Met-His$$ What is abnormal concerning these results? What might have been the problem that caused it?

Short answer

Incomplete trypsin digestion is suggested. Issues could include insufficient enzyme, inadequate reaction time, or steric hindrance.

Step-by-step solution

- Understand Trypsin Digestion

Trypsin digestion breaks a peptide chain at the carboxyl side of lysine (Lys) and arginine (Arg) residues.

- Analyze Fragments from Trypsin Digestion

The peptide fragments should end with Lys or Arg if trypsin digestion was complete. Examine the given fragments: 1. Leu-Gly-Arg 2. Gly-Ser-Phe-Tyr-Asn-His 3. Ser Glu-Asp-Met-Cys-Lys 4. Thr-Tyr-Glu-Val-Cys-Met-His

- Identify the Anomaly

From the analysis in Step 2, note that: - Leu-Gly-Arg ends with Arg (correct). - Gly-Ser-Phe-Tyr-Asn-His does not end with Lys or Arg (incorrect). - Ser-Glu-Asp-Met-Cys-Lys ends with Lys (correct). - Thr-Tyr-Glu-Val-Cys-Met-His does not end with Lys or Arg (incorrect).

- Draw Conclusions

Only two fragments end with the expected trypsin cleavage residues (Lys and Arg). The other two fragments which do not end in Lys or Arg suggest incomplete digestion.

- Identify Possible Issues

Possible issues for incomplete trypsin digestion could include: 1. Insufficient enzyme concentration. 2. Incomplete reaction time. 3. Peptide sequence resistant to trypsin cleavage due to steric hindrance.

Key concepts

Amino Acid Sequence

Determining the amino acid sequence of a peptide involves identifying the order of amino acids in the chain. Each amino acid is bonded to its neighbor by peptide bonds. The sequence is crucial for understanding the peptide's function and properties.

When analyzing a peptide, different techniques, such as enzymatic digestion and sequential degradation, are utilized to deduce the order of amino acids. The sequence provides insights into the structure and biological activity of the peptide.

Trypsin Cleavage Sites

Trypsin is an enzyme used in peptide digestion because it cleaves at specific sites, making analysis easier. It breaks peptide bonds at the carboxyl side of lysine (Lys) and arginine (Arg) residues.

During trypsin digestion, you should expect peptides to end at these cleavage points unless something interferes. For example, steric hindrance, improper enzyme concentration, or insufficient reaction time might prevent complete digestion, causing some fragments not to end in Lys or Arg.

Peptide Fragments

Peptide fragments are smaller pieces of the original peptide chain produced after enzymatic cleavage. In this exercise, the following fragments were obtained after trypsin digestion:

• Leu-Gly-Arg
• Gly-Ser-Phe-Tyr-Asn-His
• Ser-Glu-Asp-Met-Cys-Lys
• Thr-Tyr-Glu-Val-Cys-Met-His

Analyzing these fragments helps determine if digestion was complete. Properly cleaved fragments should end with Lys or Arg, indicating correct and complete trypsin action.

Edman Degradation

Edman degradation is a method used to sequence amino acids in a peptide. It removes one amino acid at a time from the N-terminus, allowing identification in order.

After trypsin digestion, Edman degradation helps further analyze peptide fragments. By determining the sequence of each fragment, scientists can reconstruct the original peptide's amino acid sequence. This method complements trypsin digestion by providing precise sequencing information.

Incomplete Digestion

Incomplete digestion refers to the scenario where not all target sites in a peptide are cleaved by the enzyme. In this exercise, two fragments did not end with Lys or Arg, suggesting incomplete trypsin digestion.

Several factors can cause incomplete digestion:

• Insufficient enzyme concentration
• Inadequate reaction time
• Steric hindrance, where the substrate physically blocks the enzyme

Recognizing incomplete digestion is crucial for accurate peptide analysis, and adjustments must be made to ensure complete digestion in future experiments.