Chemical Cleavage of Bonds

in this video, we're going to begin our discussion on the chemical cleavage of bonds. So it turns out that there's actually many different chemicals that biochemist can use to cleave the bonds within proteins. And you're definitely not expected to know all of those chemicals and the bonds that they cleave. So we're only going to focus on the more common chemicals that your professors likely going toe want you guys to know. And so the first chemical that we're gonna cover is cyanogen bromide, which can be abbreviated with C N B R and cyanogen bromide cleaves, very specific peptide bonds and the specific peptide bonds that it cleaves are the peptide bonds that are on the car boxful side of Matthias Ning, amino acid residues and so down below. In our example, it's asking us where will cyanogen bromide cleave the peptide and which will notice is we have cyanogen bromide here and we know that it could be abbreviated with C and B R. And something that I came up with that helps me remember exactly where cyanogen bromide cleaves is. If we take this, be here in the abbreviation for cyanogen bromide and we rotate it degrees counterclockwise, we can get this be to kind of look like an M. And so when we do that, it's literally telling us where cyanogen bromide cleaves it. Cleese next to Matthiasson ing residues. And so hopefully that will help you guys remember where cyanogen bromide cleaves and so notice over here on the left, what we have is a tetra peptide or a peptide with four amino acid residues represented by these four circles. Here on the residues are Alan Matheny insisting and history in. And of course, it has an end terminal end with a free amino group. And it has a C terminal end on the opposite end with a free car boxing group and notice up above. What we have is the cyanogen bromide structure, where we have a carbon triple bonded to a nitrogen and that carbon is bonded to a bro mean And so this is cyanogen bromide. And again it's asking where cyanogen bromide going to cleave the peptide. Now, we know by rotating this, uh, be here in this abbreviation that it cleans next to meth I ning residues. And so we've got to my thinning. We've got two peptide bonds around them, a thinning residue. We've got this peptide bond here and this peptide bond over here now, because cyanogen bromide starts with the letter C here. That helps me remember that it cleaves closest to the C terminal end. And so, essentially, it's gonna cleave the peptide bond that's closest to the C terminal end, which is going to be this peptide bond right here. And so what we can do is we can draw a little zigzag line through here to represent this is the peptide bond being cleaved and weaken right, cleaved underneath of it. And so, essentially, cyanogen bromide is going to split our peptide in half so that we have an al Ani Matthiasson in fragment on one side and assisting history and fragment on the other side. And so we'll be able to get some more practice with the chemical cleavage of bonds using cyanogen bromide in our next practice video. So I'll see you guys there

so the next chemical that we're going to talk about that, cleaves. The chemical bonds within proteins is the chemical agent. Hydrazine and hydrazine is used to initiate a process called Hydro's analysis. And hydrogen analysis is used to identify the C terminal amino acid residue of a peptide. And so the way that it works is that when a peptide is treated with hydrazine, it actually forms amino Aysal hydra sides with every single amino acid residue, except for the C terminal amino acid residue. So the C terminal amino acid residue is the Onley amino acid residue that will not form an amino Aysal hydroxide. And so what that means is that the C terminal amino acid residue is going to be chemically different than all of the other amino acid residues after treatment with hydrazine. And so what that means is that this free C terminal amino acid residue can be pretty easily distinguished from all of the other residues, since it's chemically different and it can be pretty easily identified. And that's exactly why we use hydrazine and Hydro's analysis in the first place is to identify the C terminal amino acid residue. So let's take a look at our example of hydrogen analysis down below and which will notices. We're starting here with a tetra peptide or ah, peptide with four amino acid residues in it. And we can quickly tell that it's a tetra peptide just by looking at the number of our groups that it has so notice that it has Ah, 1234 different our groups. And of course which will notice is that this end over here on the left is the end terminal end because it has a free amino group. And this end over here on the right is the C terminal and because it has a free car box, Late group. And so, if we take our tetra peptide here and we treat it with hydrazine, so notice Here we have the chemical structure of hydrazine being shown. Essentially, hydrazine is going to react with our peptide in a way that every single residue is going to essentially form and amino Essel hydroxide, except for the C terminal residue. So notice that the C terminal residue over here is the Onley residue that does not form an amino Aysal hydro side, and so literally you could see that there's this chemical group that's attached to the amino end of all of the residues that makes it an amino, Aysal hydro side. But the C terminal residue does not have that same group. It actually has its normal car Boxley Group. And so that is what distinguishes the C terminal amino acid residue from all of the other amino acid residues and the peptide. And since they're so chemically different, that allows us to easily identify and distinguish the C terminal amino acid residue. So one way that helps me remember how hydrazine and hydrogen analysis works is that I associate that the fact that Z is the last letter of the alphabet and that hydrazine has a Z in it. And it's one of the Onley chemicals that has a Z in it that we're going to talk about. And so hydrazine is used to identify the last residue of a peptide or the C terminal residue of a peptide. And so if you can make that association, then you'll be able to remember how hydrazine and hydrogen analysis is used to identify C terminal amino acid residues. And so this concludes our lesson on hydrogen analysis, and we'll be able to get some practice in our next video. So I'll see you guys there

So the next set of chemicals that we're going to talk about that air used to cleave specific bonds within proteins are bottom or Capito ethanol and I Oto acetate, which are used in conjunction with one another or at the same time as each other, now recall from our previous lesson videos that bottom or capital ethanol is specifically used to cleave die sulfide bonds. So let's take a look at our example down below to refresh our memories on how bottom or capital ethanol cleaves die sulfide bonds And so notice on the far left here, what we have is a single Sistine Residue and assist in residue is just when we have to sixteens that are co violently connected via a die sulfide bond, which is shown in red right here. And so we know that with the addition of Beta Moore Capito ethanol, whose structure is shown right here that the addition of Baltimore Capito ethanol will reduce the dye sulfide bond that's here and break or cleave that die sulfide bond that's linking the two sixteens. And so that results in two separate Sistine molecules where we have one Sistine molecule over here and a separate Sistine molecule over here, and they are not co violently linked via a die sulfide bond, since bottom or captain ethanol broke that die sulfide bond. However, under the appropriate conditions, these two Sistine molecules can actually reform that die sulfide bond and form the Sistine again. And so, essentially, that's what this blue arrow is all about. And so a Z you could see through the removal bottom or capital ethanol and under oxidizing conditions, we can oxidize these two Sistine to reform the dye sulfide bond. Now it turns out that we actually want to get rid of die sulfide bonds when we're looking to sequence of protein. And the reason for that is because die sulfide bonds actually interfere with the sequencing procedure. And so because they sulfide bonds interfere with the sequencing procedure, it's important that we actually break the dye sulfide bonds prior to Edmund Degradation, sequencing and recall that we mentioned Edmund Degradation sequencing when we did the overview of direct protein sequencing in our previous lesson videos when we covered the map for these next couple of lesson videos. And so we'll cover Edmund degradation, sequencing and a lot more detail and another video later in our course. But for now, what I want you guys to know is that di sulfide bonds interfere with the sequencing procedure, and we have toe have away toe break the dye sulfide bonds so that they do not interfere. And so what that means is that using bottom or capital ethanol alone is, uh may not be sufficient to permanently break the dye sulfides because there is the possibility for them to reform the dye sulfide bond. And so that's exactly where I Oto acetate comes into play. So when we use body armor, capito ethanol in conjunction with iota acetate that actually permanently breaks the dice sulfide bonds of Sistine Residues. And so the way that it works is that iota acetate will actually car Boxley methyl eight Sistine Soft hydro groups and the car boxing methylation actually prevents the re formation of the dice sulfide bonds. So let's take a look at our example down below to clear that up. So again, once we treat our die sulfide bond with bottom or capito ethanol, it will reduce those that die sulfide bond to produce to Sistine molecules that are shown here and so these Sistine molecules to prevent the re formation of the diesel five bond, we add i Oto acetate. And so you can see here that iota acetate structure is provided down below and iota acetate will will react with each of these soft hydro groups that are shown. And it will car boxy, methylated, those soft hydro groups. So notice that we have a car boxy, methylated Sistine right here and another car boxy, methylated Sistine over here. And so you can see these car box car boxy methylated sixteen's are not able to reform this die sulfide bond that's present on the far left. So you can see we have a one way arrow going from the Sistine to the car boxy, methylated sixteens. And so this allows us to prevent the re formation of the dice sulfides. And it allows us to proceed with Edmund degradation sequencing without any interference from die sulfide bonds. And so this year concludes our lesson on bottom or capital ethanol and iota acetate, and we'll be able to get some practice in our next couple of videos. So I'll see you guys there

Hey, guys, in this video, we're going to do a quick recap on the chemical agents that we covered in our most recent lesson videos. And so none of the information in this video is new information. It's all review from our older videos. And really, this entire section is just here to help refresh your guys memories and to give you a place to consolidate all of the information that you've learned. And so what we're gonna do is fill in the blanks in the chart below to recap the effects that these chemicals have on proteins. And so in the left hand column in our chart below what we have is the chemical agent and then in the right hand column. What we have is the result that the chemical agent has on the protein and notice that our chemical agents here are Our our chart is actually color coded. And so the first chemical that we have is one floor 024 die nitrobenzene, which is also abbreviated as f d n B and known as singers re agent. And so the reason that Sanders re agent or F d N B here is, uh, color coded with blue is because it's the Onley re agent in this list that actually doesn't cleave any chemical bond of a protein. And so we know that f D N B essentially, what it does is it reacts to covertly label the free and terminal amino acid residues of all poly peptide chains. And so F D N B alone doesn't actually cleave any bonds. It just co violently labels and terminal amino acid residues. But we know from our previous lesson videos that when we use F d N B, along with six Mohler hydrochloric acid, that allows us to reveal two different things from different about our proteins. The first is it reveals the end terminal residues so we can write in terminal residues. And the second thing that it reveals is the number of sub units. And so remember that none of this information here is new information. All of it is review. And this area here is for you guys to refresh your memories and take notes and consolidate the information. And so the next chemical that we have is six Mohler hydrochloric acid or six Mohler hcl, and we know that this is used to induce a complete amino acid. Hydraulics is to cleave all of the peptide bonds found in a protein and to release all of the amino acid residues as free amino acids. And we know that if we follow up the use of six Miller hydrochloric acid with techniques such as H, P, L. C or mass spectrometry that were actually able to reveal the amino acid composition. All right, so our next chemical that we have is cyanogen bromide. And we know that cyanogen bromide can be abbreviated with CNB are. And we know that by taking this b and rotating it 90 degrees counterclockwise that it tells us literally where it cleaves. So cyanogen bromide cleaves next to meth I ning residues. And so what we'll see here is that it cleaves the peptide bonds on the C terminal side. And we noticed the C terminal side because cyanogen bromide starts with the letter c here. So that helps remind us that it cleans the C terminal side of meth I ning residues. And, uh, the next chemical that we have is hydrazine and hydrazine structure is shown right here and h two n. H two and we know that hydrazine because it has the Z in it and Z is the last letter of the alphabet. Hydrazine is used to initiate hydrogen analysis and identify the last residue of a protein, and so it identifies the C terminal amino acid residue of a protein. Now, the last but not least, what we have is Beta Moore Capito ethanol and I Odo Acetate. And this one's kind of easy because we've already covered bottom or capito ethanol plenty of times in our previous lesson videos. And we know that it's used specifically to cleave die sulfide bonds to cleave or break di sulfide bonds. And so the iota acetate here is really just used to car boxy methyl it, the soft hydro groups to prevent the re formation of the dice sulfide bonds. So essentially bottom or captive ethanol and iota acetate permanently breaks the dice sulfide bonds, and we know that the dye sulfide bonds interfere with sequencing and that we're gonna need thio break any dye sulfide bonds before we move forward with the Edmund degradation sequencing. And so this here is our quick recap of the chemicals, and we'll be able to get some more practice in our next couple of videos, so I'll see you guys there