in this video, we're going to talk about a chemical called F D N B so F D N B is really just an abbreviation for a chemical called one floor 024 Die Nitro Benzene and F. D N B is specifically used to co violently label all of the free end terminal amino acid residues on all of the poly peptide chains that are present in the sample. Now F D N B is sometimes also called Sanger's re agent, and the reason is because it's named after the scientist Frederic Singer, who first used F d N b toe identify the N terminal amino acid residue of proteins. So in our example below, we're gonna talk about the effect that F. D N B 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 two different sub units, one sub unit and blue on the left and another sub unit and green on the right. And so these two sub units we know our separate poly peptide chains and each poly peptide chain is going toe have its own end 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 F, d and B. Now, when we treat our single protein here with F D N B. This pink molecule that's shown up above we know that F. D N B is going to co violently label all of the free and terminal amino acid residues on all of the poly peptide chains now, because we have to sub units in our protein. We know that we have to poly peptide chains and each poly peptide chain and terminal residue is gonna be labeled by F d N B. So when we look at the right here, which will notice is we've got the n terminal residue being shown for both of the poly peptide chains, and you can see that there indeed labeled with this F D N B molecule, and they are co violently labeled, so you can see that there is a co Vaillant bond that's linking the F. D N B molecule to the, uh, 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, uh, not affected by F d N B. So essentially, what we're saying is that this squiggly line represents the rest of this blue poly peptide chain, and this green squiggly represents the rest of the green poly peptide chain. They're just not being shown because they're abbreviated with these Quigley's here. And so essentially, the major take away from this video is that the use of F. D. N B will label Co. Violently label the in terminal residue of all of the poly peptide chains that are present. So if there's only one poly peptide chain, then on Lee, the N terminal residue for that one poly peptide chain will be labeled. But if there are three poly peptide chains, then all three and terminal residues for all three poly peptide chains will be labeled so, uh, down below, we could say the F. D N B labels all end terminal residues. So this year concludes this video, and in our next video, we're going to talk about how f d n B can be used with amino acid hydraulics ISS to reveal not only the n terminal residue but also the number of sub units that a protein has. So I'll see you guys in that video.
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So in our last lesson video, we said that F. D N B is a chemical that reacts to co violently label the free and terminal amino acid residues of all poly peptide chains. And so in this video, we're going to talk about how F D. N B is used with amino acid. Hydraulics is to reveal critical information about a protein, and so, typically, what we'll see is the F D N B treatment will be followed up by complete amino acid hydraulics issues ing six Mueller hydrochloric acid to non specifically cleave all of the peptide bond in a protein and release all the amino acids as free amino acids. Now because previously F D N B Co. Violently labeled the n terminal amino acid residues, the N terminal amino acid residues are released as Dinei Tral Final derivatives, or D N. P s. And these d MPs are pretty easily analyze via techniques such as H PLC or mass spectrometry to identify the end terminal amino acid residues that again were previously labeled by F D N B. Now, another thing the F d. N. B allows biochemist to determine is the number of sub units that a protein has. And so it turns out that the amount and the types of D MPs that air detected will indicate the number of sub units. And so if a biochemist has no idea how Maney subunits their protein has, they can use F d N B to not only reveal the n terminal amino acids, but also the number of sub units that the person has. And so, in our example below, we're going to talk about how f d N B reveals not only and terminal amino acid residues, but also the number of sub units 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 Ah protein here with Quaternary structure treated with F. D N B, will label all of the n terminal amino acid residues, and that's exactly what's being shown here. Now, if we add F six Mohler hydrochloric acid after f d. N B treatment that will induce complete amino acid, hydraulics is 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 here represent free amino acids. Now again, because we previously labeled the n terminal amino acid residues with F D N B. Those and terminal amino acid residues are released as D. N P derivatives, and so these are the D. N. P s. Now we can follow up all of this with the use of a techniques such as H PLC or mass spectrometry. Here we're using H PLC, and you can see that we have a H p H PLC chromatic Graham showed over here where we have the absorb, it's on the Y axis and the illusion time on the X axis. And by using known controls, we can reveal the amino acids that are found in our, um our protein. And so, which will notice is that some of these peaks correspond to the D. M, P, S and others correspond to the free amino acids. And so the result here is going to be the D. M. P 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, uh, Alan in here are the A And there were three alunan residues were 1 16 residue wanna Spartak acid and to glue tannic acid residues and so on. And so what you'll notice is that we have the d. N P s. That can be pretty easily detected here. So we have DNP f or DNP, phenylalanine and D N p R or D N P Argentine. And notice that the DNP F can be distinguished from regular fetal Alan means that air, not the n terminal amino acid residue. So remember that the D. M. P s are labeling the n terminal amino acid residue and also recall from up above that the amount and types of D MPs indicates the number of sub units. So because we detected two D MPs here DNP phenylalanine and DNP Argentine that tells us that, uh, two and two d MPs actually indicates to sub units and our protein and also because the D. M P s are labeling the in terminal amino acid residue. We have a DNP phenylalanine, meaning that we have a female al inning as one of our in terminal residues. And then we have DNP Argentine, which means that we have Argentine as the other end terminal amino acid residue for those two sub units. And so you can see here how f D N B can reveal not only the in terminal residues but also the number of sub units. And really, that's the main take away from this video. And so also, what's important to note is that Daniel chloride and dabs will chloride are used in a very similar way to F d N B. So sometimes you might find that your teacher or your textbook might use Daniel or dazzle chloride. But essentially, it's used in a very similar way toe label, the in terminal minnow 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 DNP and amino acid composition, that composition can actually be used to strategize. It could 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 and are later videos. But again, for now, the major result or the major take away of this video is that F D N B reveals and terminal residues and the number of sub units. So we'll be able to get some practice utilizing these concepts and 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 free Lys labeled by FDNB & an unlabeled tetrapeptide.
5 free amino acids labeled by FDNB.
5 free amino acids with Lys & Arg labeled by FDNB.
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?
The protein’s N-terminal residue is Arg & its C-terminal residue is Asp.
The protein’s N-terminal residue is Asp & its C-terminal residue is Arg.
The protein has two subunits; one with an Arg N-terminal residue and one with an Asp N-terminal residue.
Perhaps the protein has not been completely purified after all.
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.