In this video, we're gonna talk about a chemical called FDNB. So FDNB is really just an abbreviation for a chemical called 1 fluorot-four Dinitrobenzene. And 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. Now, Now, FDNB is sometimes also called Sanger's reagent and the reason is because it's named after the scientist, Frederick Sanger, who first used FDMB to identify the n terminal amino acid residue of proteins. So in our example below, we're gonna talk about the effect that FDMB has on proteins. So what you'll notice is on the far left over here, what we have is a single protein that has quaternary structure to it because it has 2 different subunits. One subunit in blue on the left and another subunit in green on the right. And so these 2 subunits we know are separate polypeptide chains, and each polypeptide chain is going to have its own n terminal end and its own c terminal end. Now notice here that we're showing the n terminal amino acid residue for both of these subunits. And the c terminal amino acid residue is not being shown because it doesn't really react with FDNB. Now 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. Now because we have 2 subunits in our protein, we know that we have 2 polypeptide chains, and each polypeptide chain N terminal residue is gonna be labeled by FDNB. So when we look at the right here, what you'll notice is we've got the N terminal residue being shown for both of the polypeptide chains. And you can see that they are indeed labeled with this FDNB molecule, and they are covalently labeled. So you can see that there is a covalent bond that's linking the FDNB molecule to the, n terminal amino acid residue. And you'll notice that we also have these squiggly lines here, and these squiggly lines just represent the rest of the protein, which is, not affected by FDNB. So, 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 squigglies here. And so essentially, the major takeaway from this video is that the use of FDNB, will label, covalently label, the N terminal residue of all of the polypeptide chains that are present. So if there's only 1 polypeptide chain, then only the n terminal residue for that one polypeptide chain will be labeled. But if there are 3 polypeptide chains, then all 3 n terminal residues for all 3 polypeptide chains will be labeled. So, down below we can say that FDMB labels all n terminal residues. So 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. So, I'll see you guys in that video.
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concept
FDNB
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Video transcript
So in our last lesson video, we said that f DNB 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 FDMB is used with amino acid hydrolysis to reveal critical information about a protein. And so typically what we'll see is that FDMB treatment will be followed up by complete amino acid hydrolysis using 6 hydrochloric acid to nonspecifically cleave all of the peptide bonds in a protein and release all of the amino acids as free amino acids. Now because previously, FDNB covalently labeled the n terminal amino acid residues, the n terminal amino acid residues are released as dinitrophenyl derivatives or DNP's. And these DNP's are pretty easily analyzed via techniques such as HPLC or mass spectrometry to identify the n terminal amino acid residues that, again, were previously labeled by FDMB. Now another thing that FDMB allows biochemists to determine is the number of subunits that a protein has. And so it turns out that the amount and the types of DMPs that are detected will indicate the number of subunits. And so if a biochemist has no idea how many subunits their protein has, they can use FDMB to not only reveal the n terminal amino acids, but also the number of subunits that the protein has. And so in our example below, we're gonna talk about how FDNB reveals not only n terminal amino acid residues, but also the number of subunits. And notice over here on the left, we have the same exact image that we have up above from our previous lesson video. And so we know that, a protein here with quaternary structure treated with FDNB will, label all of the N terminal amino acid residues. And that's exactly what's being shown here. Now, if we add f, 6 molar hydrochloric acid after FD and B treatment, that will induce complete amino acid hydrolysis to non specifically cleave all of the peptide bonds in the protein, and that will release all of the amino acids as free amino acids. And so all of these circles N terminal amino acid residues with FDMB, those N n terminal amino acid residues with FDNB, those n terminal amino acid residues are, released as DNP derivatives. And so these are the DNPs. Now, we can follow-up all of this with the use of a technique such as HPLC or mass spectrometry. Here, we're using HPLC. And you can see that we have a HP an HPLC chromatogram shown over here, where we have the absorbance on the y axis and the elution time on the x axis. And by using known controls, we can reveal the amino acids that are, found in our, our protein. And so what you'll notice is that some of these peaks correspond to the DNPs and others correspond to the free amino acids. And so the result here is going to be the DMP and the amino acid composition. And so what you'll see down below is that we have the amino acid one letter codes and the number of amino acids that were found. And so for instance, we have, alanine here, our, the a, and there were 3 alanine residues. There were, one cysteine residue, one aspartic acid, and 2 glutamic acid residues, and so on. And so what you'll notice is that, we have the DNPs that can be pretty easily detected here. So we have DNPF or DNP Phenylalanine, and DNP R or DNP Arginine. And notice that the DNPF can be distinguished from regular phenylalanines that are not the n terminal amino acid residue. So remember that the DNPs are labeling the N terminal amino acid residue. And also recall from up above that the amount and types of DNPs indicates the number of subunits. So because we detected 2 DMPs here, DMP Phenylalanine and DMP Arginine, that tells us that, 2 2 DMP's actually indicates 2 subunits in our protein. And also because the DMP's are labeling the n terminal amino acid acid residue, we have a DMP phenylalanine, meaning that we have, phenylalanine as one of our n terminal residues. And then we have DMP arginine, which means that we have arginine as the other N terminal amino acid residue for those 2 subunits. And so, you can see here how FDMB can reveal not only the n terminal residues, but also the number of subunits. And really, that's the main takeaway from this video. And so, also what's important to note is that, damself chloride and dabsyl Chloride are used in a very similar way to FDMB. So sometimes, you might find that your teacher or your textbook might use Danzil or dabsyl chloride, but essentially, it's used in a very similar way to label the n terminal amino acid residues, and it essentially reveals the same information that we have here. And so, lastly, because this technique here allows us to reveal the DMP and amino acid composition. That composition can actually be used to, strategize. It can be used to strategize the protein fragmentation prior to sequencing. And so we'll be able to better understand how we could possibly strategize protein fragmentation after we cover our protein fragmentation techniques in our later videos. But again, for now, the major result, or the major takeaway of this video is that FDMB reveals n terminal residues and the number of subunits. So we'll be able to get some practice utilizing these concepts in our next couple of videos, so I'll see you guys there.
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Problem
Problem
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.
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Problem
Problem
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.
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Problem
Problem
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.