Understanding Phenyl Isothiocyanate Reaction with Amino-Terminal Residues

Protein sequencing is a fundamental technique in molecular biology, essential for determining the order of amino acids in a protein. One of the key reagents used in this process is phenyl isothiocyanate (PITC), which reacts specifically with the amino-terminal residue of proteins. This reaction forms the basis of the Edman degradation method, which is widely used for protein sequencing.

Correct Answer: (C) P and Q

Keyphrase: Phenyl Isothiocyanate Reaction

Phenyl isothiocyanate reaction is a crucial step in protein sequencing, as it selectively reacts with the amino-terminal residue of a protein.

How Phenyl Isothiocyanate (PITC) Reacts with Proteins

1. Role of Phenyl Isothiocyanate in Protein Sequencing

Phenyl isothiocyanate is a chemical reagent used in the Edman degradation method to identify amino acid sequences in proteins. The reaction involves:

• Binding of PITC to the amino-terminal (N-terminal) residue of a protein.
• Formation of a phenylthiocarbamyl (PTC) derivative.
• Cleavage of the PTC derivative to release the modified amino acid.
• Sequential identification of the released amino acids using chromatography or electrophoresis.

2. Specificity of Phenyl Isothiocyanate Reaction

• PITC specifically targets the free amino-terminal group of a protein.
• If the amino-terminal residue is blocked, such as in N-formyl methionine, the reaction will not proceed.

Explanation of the Statements

How PITC Reaction Works in Edman Degradation

Step 1: Reaction with PITC

• PITC reacts with the free amino-terminal residue in the presence of a mild alkaline solution.

Step 2: Formation of PTC Derivative

• The reaction forms a stable phenylthiocarbamyl (PTC) derivative of the amino acid.

Step 3: Cleavage and Identification

• The PTC derivative is cleaved under acidic conditions.
• The released amino acid derivative is identified using chromatography or electrophoresis.

Why PITC Does Not Work with Blocked Amino-Terminal Residues

Importance of Phenyl Isothiocyanate in Protein Sequencing

1. Structural Analysis of Proteins

• Edman degradation using PITC allows identification of the amino acid sequence of small proteins and peptides.

2. Protein Modification Studies

• Identification of blocked amino-terminal residues helps in understanding post-translational modifications.

3. Functional Studies

• Knowledge of protein sequences provides insights into protein structure and function.

Challenges in PITC-Based Protein Sequencing

Applications of PITC in Molecular Biology

Techniques Complementing PITC-Based Sequencing

Why Understanding PITC Reaction is Important for CSIR NET and Other Exams

Protein sequencing and phenyl isothiocyanate reactions are key topics in molecular biology and biochemistry. These concepts are essential for:

• CSIR NET Life Science
• IIT JAM Biotechnology
• GATE Biotechnology
• DBT JRF

Summary

Phenyl isothiocyanate is a key reagent in protein sequencing. It specifically reacts with the amino-terminal residue of a protein, facilitating stepwise cleavage and identification of amino acids. However, blocked amino-terminal residues, such as N-formyl methionine, prevent the reaction from proceeding. This makes PITC-based protein sequencing highly specific and accurate for studying protein structure and function.

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FAQs

Q1. What is phenyl isothiocyanate used for in protein sequencing?
Phenyl isothiocyanate reacts with the amino-terminal residue, facilitating sequential cleavage of amino acids for sequencing.

Q2. Why does PITC not react with N-formyl methionine?
N-formyl methionine has a blocked amino-terminal group, preventing PITC binding and reaction.

Q3. How does Edman degradation work?
Edman degradation uses PITC to cleave and identify amino acids sequentially from the amino-terminal end of a protein.

This article was prepared with insights from Let’s Talk Academy, the top institute for life science competitive exams.