FDNB: Videos & Practice Problems

FDNB, or 1-fluoro-2,4-dinitrobenzene, is a chemical reagent commonly known as Sanger's reagent, named after the scientist Frederick Sanger. This compound is primarily utilized to covalently label the free N-terminal amino acid residues of polypeptide chains in protein samples. Understanding the role of FDNB is crucial for protein analysis, particularly in identifying the N-terminal residues.

In a protein with quaternary structure, which consists of multiple polypeptide chains, each chain has its own N-terminal and C-terminal ends. The N-terminal end is where FDNB acts, forming a covalent bond with the amino acid at this position. For instance, in a protein composed of two subunits, one represented in blue and the other in green, FDNB will label the N-terminal residues of both subunits. The C-terminal residues do not react with FDNB, making them irrelevant in this context.

When treated with FDNB, the N-terminal residues of each polypeptide chain become covalently bonded to the FDNB molecule, which can be visually represented in diagrams. The remaining structure of the protein is often depicted with squiggly lines, indicating that while the N-terminal residues are labeled, the rest of the polypeptide chains remain unaffected by the reagent.

In summary, FDNB is a powerful tool for labeling N-terminal residues across multiple polypeptide chains in a protein. This labeling is essential for further analysis, such as determining the number of subunits in a protein and identifying specific amino acids at the N-terminal position. The next steps in protein analysis may involve amino acid hydrolysis, which can provide additional insights into the protein's structure and composition.

In biochemical analysis, the use of FDNB (dinitrofluorobenzene) plays a crucial role in identifying the N-terminal amino acid residues of polypeptides. This chemical covalently labels the free N-terminal amino acids, allowing for subsequent analysis after complete amino acid hydrolysis. The hydrolysis process typically involves treating the protein with 6 M hydrochloric acid, which cleaves all peptide bonds, releasing the amino acids as free entities.

Following the FDNB treatment and hydrolysis, the N-terminal amino acids are released as dinitrophenyl derivatives (DNPs). These DNPs can be effectively analyzed using techniques such as High-Performance Liquid Chromatography (HPLC) or mass spectrometry. The chromatographic analysis provides a visual representation of the amino acid composition, where peaks correspond to both DNPs and free amino acids. For instance, if the analysis reveals DNP derivatives for phenylalanine (DNPF) and arginine (DNP R), it indicates the presence of these amino acids as N-terminal residues in the protein.

Moreover, the number and types of DNPs detected can also inform biochemists about the number of subunits within a protein. For example, if two distinct DNPs are identified, it suggests that the protein consists of two subunits, each with its respective N-terminal amino acid. This method not only elucidates the identity of the N-terminal residues but also provides insight into the protein's quaternary structure.

It is worth noting that other reagents, such as dansyl chloride and dabsyl chloride, can be utilized similarly to FDNB for labeling N-terminal residues, yielding comparable information. The amino acid composition derived from these analyses can be instrumental in strategizing protein fragmentation prior to sequencing, enhancing the understanding of protein structure and function.

In summary, the application of FDNB in conjunction with amino acid hydrolysis is a powerful technique for revealing critical information about proteins, including the identification of N-terminal residues and the determination of subunit composition.