FDNB - Online Tutor, Practice Problems & Exam Prep

FDNB, or 1-fluoro-2,4-dinitrobenzene, is used to covalently label free N-terminal amino acid residues in polypeptide chains, revealing critical information about proteins. Following FDNB treatment, amino acid hydrolysis with hydrochloric acid releases these residues as dinitrophenyl derivatives (DNPs), which can be analyzed using HPLC or mass spectrometry. This process not only identifies N-terminal residues but also indicates the number of subunits in a protein, enhancing understanding of protein structure and composition.

FDNB

In this video, we're going to talk about a chemical called FDNB. FDNB is really just an abbreviation for a chemical called 1-fluoro-4-dinitrobenzene. FDNB is specifically used to covalently label all of the free N-terminal amino acid residues on all of the polypeptide chains that are present in the sample. FDNB is sometimes also called Sanger's reagent, and the reason is because it's named after the scientist, Frederick Sanger, who first used FDNB to identify the N-terminal amino acid residue of proteins. In our example below, we're going to talk about the effect that FDNB has on proteins.

What you'll notice on the far left over here is that we have a single protein that has quaternary structure to it because it has two different subunits. One subunit in blue on the left and another subunit in green on the right. These two subunits are separate polypeptide chains, and each polypeptide chain is going to have its own N-terminal end and its own C-terminal end. Notice here that we're showing the N-terminal amino acid residue for both of these subunits. The C-terminal amino acid residue is not being shown because it doesn't really react with FDNB.

When we treat our single protein here with FDNB, this pink molecule that's shown up above, we know that FDNB is going to covalently label all of the free N-terminal amino acid residues on all of the polypeptide chains. Since we have two subunits in our protein, we know that we have two polypeptide chains, and each polypeptide chain N-terminal residue is going to be labeled by FDNB. When we look to the right here, you'll notice the N-terminal residue being shown for both of the polypeptide chains. You can see that they are indeed labeled with this FDNB molecule, and they are covalently labeled. You can see that there is a covalent bond linking the FDNB molecule to the N-terminal amino acid residue. You'll notice that we also have these squiggly lines here, which just represent the rest of the protein, which is not affected by FDNB. Essentially, what we're saying is that this squiggly line represents the rest of this blue polypeptide chain, and this green squiggly represents the rest of the green polypeptide chain. They're just not being shown because they're abbreviated with these squiggles here.

Essentially, the major takeaway from this video is that the use of FDNB will covalently label the N-terminal residue of all of the polypeptide chains that are present. If there's only one polypeptide chain, then only the N-terminal residue for that one polypeptide chain will be labeled. But if there are three polypeptide chains, then all three N-terminal residues for all three polypeptide chains will be labeled.

This here concludes this video, and in our next video, we're going to talk about how FDNB can be used with amino acid hydrolysis to reveal not only the N-terminal residue but also the number of subunits that a protein has. I'll see you guys in that video.

FDNB

So in our last lesson video, we said that FDNB is a chemical that reacts to covalently label the free N-terminal amino acid residues of all polypeptide chains. And so in this video, we're going to talk about how FDNB is used with amino acid hydrolysis to reveal critical information about a protein. Typically, what we'll see is that FDNB treatment is followed by complete amino acid hydrolysis using 6 molar hydrochloric acid to nonspecifically cleave all the peptide bonds in a protein and release all the amino acids as free amino acids. Now, because previously, FDNB covalently labeled the N-terminal amino acid residues, these residues are released as dinitrophenyl derivatives or DNPs. DNPs are pretty easily analyzed via techniques such as HPLC or mass spectrometry to identify the N-terminal amino acid residues that were previously labeled by FDNB. Another thing that FDNB allows biochemists to determine is the number of subunits that a protein has. The amount and types of DNPs detected will indicate the number of subunits. If a biochemist has no idea how many subunits their protein has, they can use FDNB to not only reveal the N-terminal amino acids but also the number of subunits that the protein has. In our example below, we talk about how FDNB reveals not only N-terminal amino acid residues but also the number of subunits. Notice on the left, we have the same image from our previous lesson video. A protein with quaternary structure treated with FDNB will label all the N-terminal amino acid residues. If we add 6 molar hydrochloric acid after FDNB treatment, it induces complete amino acid hydrolysis to nonspecifically cleave all the peptide bonds in the protein, releasing all the amino acids as free amino acids. These N-terminal amino acid residues with FDNB are released as DNP derivatives, shown here as DNPs. We follow up all of this with the use of a technique such as HPLC. We have an HPLC chromatogram shown here, with absorbance on the Y-axis and elution time on the X-axis. By using known controls, we can identify the amino acids in our protein. Some peaks correspond to DNPs and others to free amino acids. The result is the DMP and amino acid composition. We have amino acid one-letter codes and the number of amino acids found. For instance, we have alanine (A) with three alanine residues, one cysteine residue, one aspartic acid, and two glutamic acid residues, and so on. DNPs can be easily detected; for example, DNPF or DNP Phenylalanine, and DNP R or DNP Arginine. DNPF can be distinguished from regular phenylalanines that are not N-terminal amino acid residues. The amount and types of DNPs indicate the number of subunits. Since we detected two DNPs here, DNP Phenylalanine and DNP Arginine, it tells us there are two subunits in our protein. DMP Phenylalanine means that phenylalanine is one of our N-terminal residues, and DMP Arginine means that arginine is the other N-terminal amino acid residue for those two subunits. FDNB reveals not only the N-terminal residues but also the number of subunits. Finally, it is important to note that dansyl chloride and dabsyl chloride are used in a very similar way to FDNB. Sometimes your teacher or textbook might use dansyl or dabsyl chloride, but essentially, it's used in a very similar way to label the N-terminal amino acid residues, and it reveals the same information we have here. Lastly, this technique allows us to reveal the DMP and amino acid composition, which can be used to strategize protein fragmentation prior to sequencing. We will better understand how we could possibly strategize protein fragmentation after we cover our protein fragmentation techniques in later videos. For now, the major result, or the major takeaway of this video, is that FDNB reveals N-terminal residues and the number of subunits. We will be able to get some practice utilizing these concepts in our next couple of videos, so I'll see you guys there.

The peptide Lys-Leu-Leu-Arg-Phe is incubated with FDNB. After exposure to 6M HCl, the solution contains:

• A. A free Lys labeled by FDNB & an unlabeled tetrapeptide.
• B. 5 free amino acids labeled by FDNB.
• C. 5 free amino acids with Lys & Arg labeled by FDNB.
• D. Options a, b & c are all incorrect.

After purifying a protein, you react the protein with 1-fluoro-2,4-dinitrobenzene (FDNB or Sanger's reagent) then with 6M HCl. You obtain DNP-Arg and DNP-Asp. Which of the following is an appropriate conclusion of the results?

• A. The protein's N-terminal residue is Arg & its C-terminal residue is Asp.
• B. The protein's N-terminal residue is Asp & its C-terminal residue is Arg.
• C. The protein has two subunits; one with an Arg N-terminal residue and one with an Asp N-terminal residue.
• D. Perhaps the protein has not been completely purified after all.
• E. c and d.

A 660 mg sample of a protein with quaternary structure (MW=132,000 g/mol) was treated with excess 1-fluoro-2,4-dinitrobenzene (FDNB) under slightly alkaline conditions until the reaction was complete. After FDNB treatment, the peptide bonds of the protein were then completely hydrolyzed by heating it with concentrated 6M HCl. The resulting solution was found to contain 5.5 mg of DNP-Val (shown in the figure below):

2,4-Dinitrophenyl derivatives of the α-amino groups of the other amino acids were not detected. Assuming 100% yield for the reaction of FDNB with the protein, determine the number of subunits in this protein.

• A. 1.
• B. 2.
• C. 3.
• D. 4.
• E. 5.

Here’s what students ask on this topic:

What is FDNB and how is it used in protein analysis?

FDNB, or 1-fluoro-2,4-dinitrobenzene, is a chemical reagent used to covalently label free N-terminal amino acid residues in polypeptide chains. This labeling helps in identifying the N-terminal residues of proteins. After treating a protein with FDNB, amino acid hydrolysis using hydrochloric acid releases these labeled residues as dinitrophenyl derivatives (DNPs). These DNPs can be analyzed using techniques like HPLC or mass spectrometry. This process not only identifies the N-terminal residues but also indicates the number of subunits in a protein, providing critical information about the protein's structure and composition.

How does FDNB help in determining the number of subunits in a protein?

FDNB helps determine the number of subunits in a protein by labeling the N-terminal amino acid residues of each polypeptide chain. After FDNB treatment, amino acid hydrolysis releases these residues as dinitrophenyl derivatives (DNPs). The number and types of DNPs detected through HPLC or mass spectrometry indicate the number of subunits. For example, if two different DNPs are detected, it suggests that the protein has two subunits, each with a distinct N-terminal residue. This method provides valuable insights into the quaternary structure of the protein.

What techniques are used to analyze DNP derivatives after FDNB treatment?

After FDNB treatment and subsequent amino acid hydrolysis, the dinitrophenyl derivatives (DNPs) of the N-terminal amino acid residues are analyzed using techniques such as High-Performance Liquid Chromatography (HPLC) and mass spectrometry. HPLC separates the DNPs based on their chemical properties, allowing for their identification and quantification. Mass spectrometry provides detailed information about the molecular weight and structure of the DNPs. These techniques help in identifying the N-terminal residues and determining the number of subunits in the protein.

What is the role of hydrochloric acid in the FDNB protein analysis method?

In the FDNB protein analysis method, hydrochloric acid (HCl) is used for complete amino acid hydrolysis. After FDNB labels the N-terminal amino acid residues, 6 M HCl is added to nonspecifically cleave all peptide bonds in the protein, releasing all amino acids as free amino acids. The N-terminal residues, previously labeled by FDNB, are released as dinitrophenyl derivatives (DNPs). These DNPs can then be analyzed using HPLC or mass spectrometry to identify the N-terminal residues and determine the number of subunits in the protein.

What are some alternatives to FDNB for labeling N-terminal amino acid residues?

Alternatives to FDNB for labeling N-terminal amino acid residues include dansyl chloride and dabsyl chloride. These reagents function similarly to FDNB by covalently labeling the free N-terminal amino acid residues in polypeptide chains. The labeled residues can then be analyzed using techniques like HPLC or mass spectrometry to identify the N-terminal residues and determine the number of subunits in the protein. These alternatives provide similar information and are used in various biochemical applications.