Introduction to Protein Synthesis and Trypsin Digestion

Protein synthesis is a fundamental biological process where amino acids are assembled into polypeptide chains according to the genetic code. The process begins at the N-terminus and proceeds towards the C-terminus, with each amino acid added sequentially. In experimental biochemistry, researchers often use radioactive labeling—such as with 14C-labeled amino acids—to track the progress and dynamics of protein synthesis.

Trypsin digestion is a widely used technique in proteomics and biochemistry to break down proteins into smaller peptide fragments. Trypsin cleaves peptide bonds specifically at the C-terminal side of lysine (K) and arginine (R) residues, generating a predictable set of peptide fragments368. This allows for detailed analysis of protein structure, synthesis, and post-translational modifications.

Experimental Setup

Consider a hypothetical protein with four equally spaced trypsin-sensitive sites, resulting in five peptide fragments upon digestion: A1, A2, A3, A4, and A5. In this scenario, A2 represents the N-terminal fragment, and A5 is the C-terminal fragment. This arrangement is essential for understanding how synthesis and labeling dynamics affect the distribution of radioactive label among the peptide fragments.

At time t = 0, all 20 amino acids are added, each labeled with 14C, and protein synthesis is initiated. At t = 4, the full-length protein is completely synthesized. If synthesis is stopped at t = 1—when only a portion of the protein has been synthesized—and the incomplete protein is digested with trypsin, the distribution of the 14C label among the peptide fragments will reflect the synthesis kinetics.

Understanding Synthesis Kinetics and Label Incorporation

During protein synthesis, the ribosome adds amino acids one by one, starting from the N-terminus and moving toward the C-terminus. If synthesis is stopped early (at t = 1), only the N-terminal portion of the protein will have been synthesized and labeled with 14C. The C-terminal region will remain unsynthesized and thus unlabeled.

Given that A2 is the N-terminal fragment and A5 is the C-terminal fragment, at early synthesis times, the N-terminal region (A2) and the adjacent regions will be more heavily labeled than the C-terminal regions. However, the question asks which peptide will have the maximum 14C label—not just whether the N-terminal or C-terminal fragment is labeled.

Trypsin Digestion and Peptide Fragment Analysis

Upon trypsin digestion, the partially synthesized protein will be cleaved at the four trypsin-sensitive sites, yielding fragments A1, A2, A3, A4, and A5. Since synthesis proceeds from the N-terminus to the C-terminus, the fragments closest to the N-terminus will be synthesized first and thus will have the highest probability of being fully labeled.

However, the question specifies that A2 is the N-terminal fragment and A5 is the C-terminal fragment. This labeling implies that A1 is not part of the main protein sequence but may be a leader or signal peptide, or possibly a mislabeling in the question (since typically, the N-terminal fragment of a protein is A1 if the numbering starts from the N-terminus). However, based on the question’s wording, we must accept that A2 is the N-terminal fragment of the main protein.

Given this, at early synthesis time (t = 1), the N-terminal fragment (A2) and the immediately following fragment (A3) will be synthesized and labeled. The C-terminal fragments (A4 and A5) will not yet be synthesized and thus will not be labeled.

Determining Which Peptide Has Maximum 14C Label

To determine which peptide will have the maximum 14C label, consider the following:

• A2: N-terminal fragment, synthesized first, fully labeled.

• A3: Next fragment after A2, partially synthesized and labeled at early time (t = 1).

• A4 and A5: Not yet synthesized at t = 1, hence not labeled.

• A1: If A1 is a leader peptide or not part of the main sequence, it may or may not be labeled depending on experimental design. However, in the context of this question, if A2 is the N-terminal fragment of the main protein, A1 may not be part of the main synthesis or may be pre-existing.

Given that synthesis is stopped at t = 1, and only a portion of the protein is synthesized, the A3 fragment—being the first fragment after the N-terminal fragment (A2) that is part of the main protein synthesis—will be the one that is most heavily labeled at this early stage. This is because, at t = 1, the ribosome will have just started synthesizing into the A3 region, and the A2 region will already be complete but not necessarily the only one being labeled if the labeling is uniform and the synthesis is continuous.

However, the question’s phrasing is ambiguous about the exact relationship between A1, A2, A3, A4, and A5 in terms of synthesis order and labeling. If A2 is indeed the N-terminal fragment of the main protein, and synthesis proceeds into A3 at t = 1, then A3 would have the most newly incorporated label if the ribosome is actively adding amino acids into A3 at the time of stopping synthesis. But if A2 is fully synthesized and A3 is only just beginning, the label may be distributed between A2 and A3, with A2 being fully labeled and A3 partially.

Given standard experimental practice and the most likely interpretation—where synthesis has proceeded just beyond the N-terminal fragment (A2) into the next fragment (A3) at t = 1—A3 will have the maximum 14C label because the ribosome is actively adding labeled amino acids into A3 at the moment synthesis is stopped, while A2 is already complete and A4/A5 are not yet synthesized.

Key Concepts and Keywords

• Protein synthesis: The process of assembling amino acids into a polypeptide chain.

• Trypsin digestion: Enzymatic cleavage of proteins at lysine and arginine residues, producing peptide fragments368.

• 14C labeling: Use of radioactive carbon to track amino acid incorporation during synthesis.

• Peptide fragments: The smaller pieces of protein produced by trypsin digestion.

• N-terminal and C-terminal fragments: The ends of the protein sequence, with synthesis starting at the N-terminus.

• Synthesis kinetics: The rate and order of amino acid addition during protein synthesis.

• Radioactive labeling: Technique to monitor synthesis and degradation of biomolecules.

• Proteomics: The large-scale study of proteins, often involving trypsin digestion and mass spectrometry38.

• Missed cleavages: Incomplete digestion at trypsin-sensitive sites34.

• Partial synthesis: Stopping synthesis before the protein is complete, used to study synthesis dynamics.

Conclusion and Correct Answer

Based on the synthesis kinetics and the arrangement of trypsin-sensitive sites, when synthesis is stopped at an early time (t = 1), the peptide fragment that will have the maximum 14C label is A3. This is because, at this stage, the ribosome is actively adding labeled amino acids into the A3 region, while the N-terminal fragment (A2) is already complete and the C-terminal fragments (A4 and A5) are not yet synthesized.

Correct Answer: (1) A3

This article provides a comprehensive explanation of the experimental scenario, the role of trypsin digestion in protein analysis, and the rationale behind the correct answer, using all relevant keywords for optimal SEO performance.