Pearson+ LogoPearson+ Logo
Start typing, then use the up and down arrows to select an option from the list.

Biochemistry

Learn the toughest concepts covered in Biochemistry with step-by-step video tutorials and practice problems by world-class tutors

5. Protein Techniques
1

concept

Peptidases

clock
7m
Play a video:
Was this helpful?
in this video, we're going to begin our discussion on Pep. Today's is so by now you guys have probably picked up that pep. Today's is are just enzymes, and we know that because it ends in A S E and anything that ends in a C. It's a good indicator that it's probably an enzyme and pep. Today's are indeed enzymes that selectively catalyzed the hydraulics ISS a very specific peptide bonds. And really, that's just a fancy way for saying that peptides is our enzymes that cleave or break down very specific peptide bonds. And we know that because from our previous lessons, we know that hydraulic sis is just the process of breaking down peptide bonds with the treatment of water. Now it turns out that there are actually many different pep today's is that exist, and you're definitely not expected to know all of those pep today's is, But there are very specific peptide. Is is that your professor is likely going toe want you guys to know, and two of those peptide aces include Trips in and Kimo trips and and that's because trips and and camo trips and are both biologically relevant. Peptide aces of our digestive systems. And so they help us break down proteins found in foods down into smaller peptide fragments that ourselves can use. And so moving forward we're first going to focus on trips in and then in a different video will focus on Kimo trips in. And so trips in turns out to be one of the more specific peptide aces because it on Lee Cleaves peptide bonds on the car boxful side of both lysine and arginine, amino acid residues and recall that listens. One letter code is okay. And Argentines. One letter code is an art. And by the way, that's exactly what we meant by specific in our definition of pep. Today's is so pep. Today's is Don't just cleave random peptide bonds. They cleave very specific peptide bonds next to very specific amino acid residues. And so trips in Will Onley cleave the peptide bonds on the car boxful side of both listen and Argentine amino acid residues Now down below. In our example, we're gonna talk about a method that I came up with. That helps me remember where trips in does its peptide bond splitting, and so hopefully that will help you guys remember it, too. But before we actually get to our example, there's something very important that you guys should note. And that's the fact that cleavage of a peptide bond by a pep titties is typically blocked or inhibited for many peptide aces if pro lean if a pro lien amino acid residue is involved in the peptide bond, and so if a parolee residue is involved in the peptide bond, it's likely that that peptide bond is not going to be cleaved because it's gonna be blocked or inhibited from being cleaved. And we'll be able to see an example of this down below and are examples well, so in our example, it's asking us where will trips in do its peptide bond splitting? And this is definitely not a formal way to be able to ask this question. Ah, more formal way would be where will trips into its peptide bond cleavage. But the reason we're asking it like this is because trips in does it split in like a nights sword or after listening and Argentine and notice on the right. Over here, what we have is the amino acid structure for lysine and for Argentine and recall way back in our previous lessons when we first introduced the are groups for all of the amino acids that license and Arjun Ng are the Onley two amino acids of all 20 that have features of a night's ward. And so remember that the pneumonic to help us remember these amino acids is just dragons eat nights riding horses where the K represents lie scene and they are here represents Argentine. And because the K here in our pneumonic is for a night listens, our group really looks like a night's ward. And so you can see that license. Our group is pretty extended here, just like a night's ward is pretty extended. And just like a knight sword is used to poke somebody. We can count the number of ends that are one letter code has that we could try to poke somebody with. And so listen has 1234 point ends that we could try to poke somebody with. And that tells us that there's a four carbon start to our chain in our our group one tube 34 and then at the very end of our, um, our group, there's an amino group now for lights for Argentine notice that it only has three pointy ends that you could really try to poke somebody with. And so that tells us that it only has a three carbon start to its chain 123 and then at the bottom. It has a triangular nitrogen structure shown here. And so again, these are the two amino acids that have features of a night's ward. And because knights wards are used for poking things and for splitting things in half just like this, um, nights were to split in this watermelon in half trips and does it splitting like a night's ward or after listening and Argentine. And so, to get back to our question, where will trips and do its peptide bond splitting notice? We have this tetra peptide here with four amino acid residues, and it's gonna have an end terminal end with a free amino group and a C terminal end on the opposite side with a free car box Elite group. And here we have the enzyme trips and being shown, and we're trying to to figure out where is it going to do its peptide bond splitting well it's gonna do it, splitting it models. It's splitting after a night's ward. So after licensing and Argentine amino acid residues, so looking for lysine and arginine noticed that we have both of these residues. So it's going to cleave after both of these two residues, so that means it's gonna cleave on the car boxful side over here. Now, one thing to keep in mind is the point that we made earlier that if a pro lean amino acid residue is involved in the peptide bond that's supposed to be cleaved, that peptide bond is gonna be blocked or inhibited from being cleaved. And so notice down below that we have a pro lean amino acid residue here, and this protein amino acid residue is involved with the peptide bond that's supposed to be cleaved. Which means that this peptide bond here is actually going to be blocked and inhibited from being cleaved, meaning that it won't be cleaved. And the Onley peptide bond that will be cleaved. Is this one peptide bond shown here in red. So what we can do is write cleaved above this and then for this peptide bond here, what we could do is write blocked because per lien is blocking the cleavage of this peptide bond and so essentially, uh, trips and is going to split our peptide here in half where we're gonna have an AL inning Listen, fragment on the left here and an Argentine pro lean fragment on the right. And so, in our next video, we'll be able to get some more practice with trips in and the cleavage of peptides and proteins. So I'll see you guys in that video.
2
Problem

Draw out each of the peptide fragments that would be generated if the peptide is treated with trypsin.

Was this helpful?
3

concept

Peptidases

clock
5m
Play a video:
Was this helpful?
So now that we've covered trips and in this video, we're gonna focus on Kimo trips in. And so chemo trips in is different than trips and and the respect that it actually has. A preference for which amino acid residues that it recognizes for cleavage and so chemo trips and actually prefers breaking the peptide bonds on the car boxful side of aromatic amino acid residues and recall that the three aromatic amino acid residues are funeral Alan Ing, Tyrus ing and trip to fan or fat young whippersnappers. And so, really, you can think of trips in and Kimo trips and as being brothers and because they are brothers, they both cleave on the car boxful side of the residues that they recognize for cleavage. Now, trips in is actually the older, more responsible brother. And so it's really uptight. Any Onley does its peptide bond splitting like a night's ward, so trips and splitting is like a night's ward, and it cleaves Onley after lice in an Argentine. Now Kimo trips and on the other hand, is the younger brother, who's a lot more chill and a lot more relaxed and so, unlike trip sins, very specific uptight cleavage right after Lacey and Arjun Ing Kimo trips and who's a lot more relaxed over time, it will actually slowly cleave other residues other than the aromatic amino acid residues and those other residues that it will actually cleave are losing and Matthiasson ing residues. And so down below. We have an image to help you guys remember how kinda trips and does its cleavage. And so Kimmo trips. And I really like to think of it as Kimo and trips on. And really, you can think of kind of trips and as being a very relaxed Jamaican who's saying, Come on trips. And so Kimmo Trips in is come on trips in and he's saying, Free your words like me. And really, what he's trying to say is trips. And you don't always have to be so uptight and cleave Onley after these licensing and Argentine residues have your preferences and then over time, slowly cleave other residues. So relax. Don't be so uptight. Come on, trips and free your worries like me have a puff and relax. And so the free your worries, like me, is really the acronym that's gonna help us remember which amino acid residues, kinda trips and Cleaves so you could see the free F years for Fino Alla nine wise retire scene ws for trip to fan and then the Like Me Loosen and Meth I Inning. And so the reason it's color coded like this is because the ones that Aaron Red here are the ones that kind of trips and has a preference for, and those are the ones that it will cleave first and then slowly over enough time. It will also begin to cleave other residues like losing and meth I ning. And so, if you can remember, uh, free your words like me, you'll remember the residues that kind of trips and recognizes for cleavage. Now over here, which you'll notice is what we have is another tetra peptide or ah, peptide with four amino acid residues in it. And, of course, it's gonna have its n terminal end with a free amino group and its C terminal end with a freak, uh, car box Elite group. And because of our pneumonic here, free your worries like me, we can clearly see that there's only one amino acid residue that it will recognize for cleavage and that is female Allen in for the free here. And so the other residues are not ones that it recognizes for cleavage. And because again, Kima trips in is the younger brother of trips, and they both cleave on the car boxful side. And so it's going to cleave after final Allen in right at this position here. And so essentially, what's gonna happen is we're going to get these two fragments of fragment here with glazing and final Alan ing and another fragment over here with a winning and searing. And so one thing that's important to note moving forward in our practice problems is that unless it's otherwise indicated, we're first going to assume that kinda trips is on. Lee going to cleave. It's preferred residues, and again, it's preferred Residues are the aromatic amino acid residues, So those are female Allan E. Tyrosine and trip to fan. So that's very important to keep in mind for our practice problems moving forward. And so really, the Loosen and Mathai inning here are Onley gonna be cleaved at a very slowly if enough time is provided. So hopefully this helps you guys remember how common trips and does it cleavage, and we'll be able to get some practice in our next video so I'll see you guys there
4
Problem

Draw out the resulting peptide fragments that would be generated if the peptide is treated with chymotrypsin.

Was this helpful?
5

concept

Peptidases

clock
5m
Play a video:
Was this helpful?
So now that we've covered both trips in and Kimo trips, and in this video, we're going to talk about some other relevant peptides is that your professor may or may not expect you guys to memorize. And so in our chart below, we have a list of these relevant peptide aces and the specific peptide bonds that they hide relies, or the specific peptide bonds that they break or cleave now something that we don't want to forget from our previous lesson videos is the fact that peptides bond cleavage can actually be inhibited if a pro lean, amino acid residue participates in the peptide bond that's supposed to be cleaved. And so this actually applies for all of the peptides that are listed in our chart below. So that's something important to keep in mind as we move forward. Now, in our example below, we've got this chart with three different columns and the far left column. We have the name of the pep Today's in the middle column. We have, uh, were indicating whether the peptide bond that's cleaved is on the n terminal side or the C terminal side of the residue, and then on the far right column We have the amino acid cleavage site or the exact amino acid that the pep today's recognizes for cleavage and which will notice is that our chart here is color coded, and all of these peptides is air colored in yellow because they all cleave peptide bonds that are on the C terminal side off the residue that they recognize for cleavage. So in this column here, we can put the C terminal side for all four of these peptides is now specifically for trips, and we know from our previous lesson videos that trips and splitting is like a night's ward took Cleaves after lysine and arginine amino acid residue. So licensing and Argentine now Kimo trips in is essentially like trip sins younger brother. And so they both cleave the C terminal side of the peptide bond or the peptide bond on the C terminal side but kinda trips and is a more relaxed younger brother. So it actually has preferences for what amino acids it recognizes for cleavage, and so it actually prefers aromatic amino acid residues and the aromatic amino acid residues are, you know, Alan entire scene and trip to fan. But It will also very slowly cleave other residues that include losing and meth inning. And so remember the pneumonic to help us memorize the residues that kinda trips and recognizes for cleavage is free. Your worries like make. And so the rest of these peptides is down below we have not yet talked about, but again, thes could be peptides is that your professor may expect you guys to memorize. So the best way to find out is to, of course, just ask your professor. And so although we don't have, uh, memory tools for all of these, we do have some important key features to take note of. And so the last days, cleaves the C terminal peptide bones, and it has a preference for amino acid residues that have small neutral are groups. And so that pretty much includes most of the amino acids that fall into our hydrophobic amino acid group. And recall that are hydrophobic amino acid group was Gavin limp. And so, essentially the Onley two residues that are not included uh, that are not recognized by a last days, um that are in this group are meth. I inning and paroling soma thinning and paroling are not part of this group, so notice we have the Gavle. But the M and the P, the methane and polling are missing. And so that's something interesting to keep in mind. And then it also recognizes Searing. Now throbbing also will cleave peptide bonds on the C terminal side, but it will Onley recognize Argentine amino acid residue, so it's pretty much just like trips and, um, except it does not recognize licensing it on. Lee recognizes Argentine. Now Pepsi in is color coded in a blue color because it actually cleaves peptide bonds on the end terminal side off the residue that it recognizes for cleavage. And what you'll see is that Pepsi in is essentially recognizing the same residues as Kimo trips in. And so it recognizes the aromatic residues as well as loosen. And really, the one that it's missing is meth. I need it does not cleave Metheny and Soma thinning is missing. Soma thinning is not present and Pepsi in or in ah, last day. So my thinning is missing from both of these, so that's something interesting to keep in mind. And then our last pep today's that's listed here is car. Boxy pep. Today's A and the reason that it's color coded all by itself is because car boxy pep today's a will on Lee cleave off the C terminal residues. And so it also cleaves the end terminal peptide bond. But it will Onley cut off the C terminal residue, so the residues that are at the very, very end off the poly peptide chain. And so it will cleave off the C terminal residues by cleaving the end terminal bond and the Onley C terminal Residues that it will not cleave are Argentine, lysine and paroling. And so again, we'll be able to get some practice. Utilizing these peptides is moving forward in our practice problems, so I'll see you guys in those videos.
6
Problem

What would the resulting peptide fragments be if the following peptide were treated with excess Pepsin? 

                                H—E—L—P—M—E—P—L—E—A—S—E

7
Problem

Which is the expected result of chymotrypsin cleavage of the following peptide? 

                            Lys—Gly—Phe—Thr—Tyr—Pro—Asn—Trp—Ser—Tyr—Phe

8
Problem

You perform multiple tests to derive the amino acid sequence of a purified peptide (see results below). Which of the following peptides listed best represents the sequence of the unknown peptide?

9
Problem

A) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion exchange or size-exclusion chromatography be most appropriate for separating the fragments? Explain. 

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.


B) Suppose the same peptide was digested with chymotrypsin. Which would be the optimal separation technique? Explain.

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

Was this helpful?
10
Problem

A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly)2, (Phe)2, His, Thr, Met.  The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC.  When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered.  Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys.  2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide.  Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide.  The tetrapeptide was composed of (Lys)2, Phe, and Gly.  The native sequence was determined to be:

*Recall: Pepsin cleaves N-terminal peptide bond of F, Y, W & L residues.*

11
Problem

The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all and more than one reagent may be suitable for a given purpose.

Was this helpful?
Divider