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 that are 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.
Types of Experiments
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 in 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, which has led 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. Edman Degradation (1956): He developed the Edman degradation method, which involves stepwise removal of amino acids from the N-terminus of a protein.
DNA Sequencing (1975): Sanger adapted his Edman degradation technique for DNA sequencing, known as the Sanger sequencing method. DNA Polymerase Chain Reaction (1983): Sanger played a crucial role in developing the PCR technique, which amplified DNA fragments for sequencing.
Main Concepts:
Amino Acid Sequencing: Sanger's techniques enabled the determination of the sequence of amino acids in proteins and DNA. Chain-Termination Sequencing: The Sanger sequencing method involves terminating DNA synthesis at specific nucleotide sites, allowing the sequence to be deduced.
* Genome Sequencing: His methods paved the way for large-scale genome sequencing projects, including the Human Genome Project.
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.
Frederick Sanger's Contribution to Protein Sequencing
Experiment: Edman Degradation
Step-by-Step Details
1. Label the N-terminus of the protein: Treat the protein with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) derivative at the N-terminus.
2. Cleavage: Use acid hydrolysis to cleave the PTC residue, releasing a thiazolinone derivative of the first amino acid.
3. Extraction: Extract the thiazolinone derivative into ethyl acetate.
4. Conversion: Treat the thiazolinone residue with anhydrous hydrogen fluoride (HF) to convert it into a phenylthiohydantoin (PTH) derivative.
5. Identification: Subject the PTH derivative to HPLC or thin-layer chromatography to identify the first amino acid.
Key Procedures
PITC Labeling:This allows selective labeling of the N-terminus without affecting other functional groups. Acid Hydrolysis: Cleavage of the PTC residue ruptures the peptide bond, releasing the first amino acid.
HF Conversion:The thiazolinone derivative is unstable and requires conversion to the more stable PTH derivative for identification. HPLC/TLC Identification: These techniques separate and identify the PTH derivatives based on their chemical properties.
Significance
Edman degradation revolutionized protein sequencing, allowing scientists to determine the amino acid sequence of proteins. It has 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.