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A topic from the subject of Contributions of Famous Chemists in Chemistry.

  • 11 months ago
  • 6 min read
Frederick Sanger's Contribution to Protein Sequencing
Introduction

Frederick Sanger was a British biochemist who made significant contributions to the field of protein sequencing. His work laid the foundation for understanding the structure and function of proteins, which are essential molecules for life.

Basic Concepts
  • Proteins: Large molecules composed of amino acids linked together in a specific sequence.
  • Protein sequencing: The process of determining the order of amino acids in a protein.
  • Peptide: A short chain of amino acids.
  • Amino acid analyzer: An instrument that separates and identifies individual amino acids.
Equipment and Techniques
  • Edman degradation: A chemical method for sequencing proteins by removing one amino acid at a time from the N-terminus (the end of the protein with a free amino group).
  • Phenylisothiocyanate (PITC): A reagent used in Edman degradation that reacts with the N-terminal amino acid and forms a phenylthiocarbamoyl (PTC) derivative.
  • High-performance liquid chromatography (HPLC): A technique used to separate and identify the PTC derivatives.
Sanger's Methods

Sanger developed two main methods for protein sequencing:

  • N-terminal sequencing: Determines the sequence of amino acids from the N-terminus.
  • C-terminal sequencing: Determines the sequence of amino acids from the C-terminus (the end of the protein with a free carboxyl group).
Data Analysis

The results of protein sequencing are typically displayed using a one-letter code for each amino acid. For example, the sequence of the first five amino acids of the protein hemoglobin is:

Val-Leu-Ser-Pro-Ala

Applications
  • Identification of proteins: Protein sequencing can be used to identify proteins based on their amino acid sequence.
  • Understanding protein structure: The sequence of amino acids determines the structure and function of a protein.
  • Diagnosis of genetic diseases: Protein sequencing can be used to identify mutations in genes that cause genetic diseases.
  • Drug development: Protein sequencing can be used to design drugs that target specific proteins.
Conclusion

Frederick Sanger's contributions to protein sequencing revolutionized the field of biochemistry. His methods enabled scientists to determine the structure and function of proteins, leading to significant advances in medicine, biology, and other fields.

Frederick Sanger's Contribution to Protein Sequencing
Introduction:
Frederick Sanger was a pioneering biochemist who revolutionized protein sequencing. His groundbreaking techniques laid the foundation for modern molecular biology.
Key Points:
Insulin Sequencing (1953): Sanger determined the amino acid sequence of insulin, becoming the first person to decipher a protein's structure. This achievement earned him his first Nobel Prize.
Edman Degradation (1956): He developed the Edman degradation method, which involves the stepwise removal of amino acids from the N-terminus of a protein. This technique, while not as impactful as his sequencing work, was a significant advancement in protein chemistry.
DNA Sequencing (1975): Sanger adapted his principles to develop a method for DNA sequencing, known as the Sanger sequencing method (or chain-termination method). This earned him his second Nobel Prize.
DNA Polymerase Chain Reaction (PCR) - Clarification: While Sanger didn't *develop* PCR, his work on sequencing laid the groundwork for its efficient application and widespread use in DNA analysis. Kary Mullis is credited with the invention of PCR.
Main Concepts:
Amino Acid Sequencing: Sanger's techniques enabled the determination of the sequence of amino acids in proteins, revealing their primary structure and providing clues to their function.
Chain-Termination Sequencing: The Sanger sequencing method involves terminating DNA synthesis at specific nucleotide sites using dideoxynucleotides, allowing the sequence to be deduced by analyzing the lengths of the resulting DNA fragments. This is the key principle behind his DNA sequencing method.
Genome Sequencing: His methods paved the way for large-scale genome sequencing projects, including the Human Genome Project, transforming our understanding of genetics and disease.
Impact:
Sanger's contributions revolutionized biochemistry and molecular biology. Protein and DNA sequencing became vital tools for understanding gene structure, function, and disease. His work laid the foundation for advancements in genetics, medicine, and biotechnology. He is considered one of the most influential scientists of the 20th century.
Frederick Sanger's Contribution to Protein Sequencing
Experiment: Edman Degradation

Step-by-Step Details

  1. Labeling: Treat the protein with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) derivative at the N-terminus.
  2. Cleavage: Use anhydrous acid (e.g., trifluoroacetic acid) to cleave the PTC residue, releasing a thiazolinone derivative of the first amino acid.
  3. Extraction: Extract the thiazolinone derivative into an organic solvent (e.g., ethyl acetate).
  4. Conversion: Convert the unstable thiazolinone derivative into a more stable phenylthiohydantoin (PTH) derivative using acid (e.g., aqueous acid).
  5. Identification: Identify the PTH-amino acid using techniques such as high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).
  6. Repeat: Repeat steps 1-5 to sequentially determine the amino acid sequence. The process is repeated on the shortened peptide after each cycle.

Key Procedures and their Significance

  • PITC Labeling: This allows selective labeling of the N-terminus without affecting other functional groups, ensuring that only the first amino acid is modified.
  • Acid Cleavage: Cleavage of the PTC residue specifically breaks the peptide bond at the N-terminus, releasing the first amino acid as a derivative.
  • Conversion to PTH: The thiazolinone derivative is unstable and readily cyclizes to form a more stable phenylthiohydantoin (PTH) derivative suitable for identification.
  • HPLC/TLC Identification: These chromatographic techniques are used to separate and identify the PTH-amino acid based on its chemical properties, revealing its identity.

Significance

Edman degradation revolutionized protein sequencing, allowing scientists to determine the amino acid sequence of proteins. This has had significant applications in various fields, including biochemistry, biotechnology, and medicine. The method has been automated and improved over time, making it a widely used technique in protein analysis. Its development earned Frederick Sanger a Nobel Prize.

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