1

concept

## Isoelectric Point of a Peptide

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in this video, we're gonna talk about how to calculate the YSL electric point of a peptide. So recall from our previous lesson videos on the is electric point that the is electric point or the P I calculation is always just gonna be the midpoint or the average between the two p. K. S for the two ionization involving the neutral species. And so all this is saying is that whenever we're calculating the ISO electric point, we're only gonna average two p k s regardless of how many p K s were given. And the same is gonna apply for calculating the is electric point of a peptide. And so, before we get to our example below, there's really two ideas that I really wanna emphasize. And the first is a similar idea to the one that we talked about when we were calculating the net charge of a peptide. And this is the idea that the is electric point and the net charge of a peptide can both on Lee be estimated. That can on Lee be estimated. And this is because of the unique micro environment that each residue has in a peptide. And so the troop EI values can Onley be experimentally determined. And because we know that the micro environment is capable of shifting the PK a values of residues, you need to make sure that you're using the correct set of PK A values specifically the set of PK a values for amino acid residues and not for PK is of free amino acids. And so, if you're professor uses the same set of P. K s for amino acid residues and free amino acids, then you're good to go, and you don't have to worry about this. But just in case you should double check to make sure that your professor doesn't want you to use a specific set of P. K s for amino acid residues. And so when we're doing our practice problems, we're gonna be using a specific set of PKs for amino acid residues. And so the second idea that I really wanna emphasize is just this idea that the ISO electric point of a peptide follows similar steps for calculating the ISO electric point of amino acids with Ayan Izabal are groups. And so this little chart here, this little diagram might look familiar to you because it's really the same one from our previous lesson. So let's take a look at the four steps that we need to follow to calculate the ice electric point of a peptide. And again, the first step is just gonna be to know to know how the amino acids are groups ionized and really all this is saying is that we need to know the seven amino acids with ionized herbal are groups and recall that are pneumonic to help us remember those seven amino acids is just yucky crazy dragons eat nights riding horses. And so if you know this pneumonic and how it works, then you'll know those seven amino acid with ionized able are groups and you'll be set to go with step number one. Now step number two is going to be to order the PK a values from the smallest to the greatest. And so here we're showing Onley four p k a values. But regardless of how many PK A values there are, there could be five. There could be six. There could be eight. However many there are. You're gonna wanna, um, order them all from the smallest to the largest and so step Number three is going to be to determine the net charge of predominant structures at any pH between each pair of adjacent P. K s or neighboring P. K s. And so what you can see down here is that we've got our four p. K s ordered from smallest the greatest. And then we have these, uh, ph range is in between the p k s that we're going to check for the predominant structure. And so we have We can either imagine the predominant structure, so we don't need to draw it out unless it tells us to. Or if you're still working on, um, if you're having trouble imagining it, you can always draw the predominant structures. But the whole point is just to determine the net charges of the predominant structures. And so we've got these ranges in between each PK that we need to check, and so we would check each one of these. And so the way that you determine the net charge again of the predominant structure is by comparing the pH of the solution that we choose to the p k A values of all the ionized able groups and so Of course, the last step is going to be the to calculate Theis Electric point just by averaging the two p. K a values. So it's always just gonna be, too. It's gonna be the two p k a. Values that sandwich the ph where the predominant structure has a neutral net charge. So again, the is electric point is gonna be the pH where the structure is going to have a neutral net charge or a net charge of zero. And so whatever to PKs, they are. So if it were this one and this one, you would average these two p. K s. If this structure here had a neutral net charge. If it wasn't these, those two PKs, If it was this PK and this PK because this structure had a neutral net charge, then you would average these two p. K. S here. So it's all about where do you find that neutral net charge? And so let's go ahead and move on to our example. And in this example, it says toe estimate Theis Electric point for the following peptide d g E, which is a Spartak asset glazing and glue, tannic acid and so all we're gonna do here is consider all of the ionized able groups. So we don't really care about any of the other groups that are not ionized Well, because they're not going to contribute to the net charge. And so if they don't contribute to the net charge, then it's not going to contribute to the is electric point. And so, for D. G and E Weaken rewrite it over here. And so we basically got a few ionized groups that we need to consider. We know that the first amino acid residue in a chain is gonna have an eye on Izabal amino groups. So there is an invisible amino group that we need to consider now a Spartak acid is gonna be one of our negatively charged acidic amino acids are groups, so we know that it's our group potentially could have a negative charge, So we'll also leave it as a blank here to consider its charge. Now Glisten does not have an ionized Volare group. It's not one of our seven amino acids with invisible our group, so we don't care about it's our group and it doesn't have any ionized herbal, amino or car box of groups because it's an internal amino acid residue. Now glue tannic acid over here does have a nine izabal, our group. That's potentially negative. Um, eso we're going to consider that and then also because it's the last residue in the chain, it also has a potentially negative, um, car boxful group. And so all we need to do is, uh now that we've done step number one, we know how these amino acids are groups ionized, and we've even, um, highlighted them like this to consider their organizations. Step number two is gonna be the order The P. K s for all four of these ionized able groups in order from smallest to largest. So now we're going to use are P k A set over here for amino acid residues, and we'll start with the amino group over here, and the amino group is going to be the n terminus. And so notice that the N terminus has a PK of eight and the PKK of eight is when we compared to all of the other PK, is that we're gonna be looking at We have glue tannic e for glue. Tannic acid has a PKF 4.1. We have a Spartak acid, which is the D here has a PK of 3.9 and then we also have the car boxful group and the car boxful group is the C terminus up here, which has a PK of 3.5. So we have our four p. K s. We need to order them from smallest to largest, and so the smallest one is the P k of the C terminus. So we'll put 35 over here, and we'll put car boxful above it to remind us it's the car boxing group. The next one is a Spartak acid. So a Spartak acid is 39 and this is well put SP for a Spartak acids are group. The next one is going to be glue tannic acid, which is 4.1. So we'll put 4.1 in a G l u for glue, tannic acid. And then, of course, our last one is going to be the end terminus, which is, um, 8.0. And so this is for the amino group. And so again, all we need to do is choose any any range we can choose this range here or this range here or this range here. It doesn't matter. You can start with anyone that you want. Let's start with one on the left since we normally do things from left to right anyways, so lets choose this range right here. And we can pick any pH that we want as long as it falls into this range. As long as it falls between these two PK A values. So let's just choose, I don't know, 3.6 ph of 3.6. And so all we need to do is imagine the predominant structure at ph 3.6 and determine the net charge of the predominant structure at Ph. 3.6. And so all we need to do is compare the Ph Thio each of the PKK. So let's start with the amino group. So with the amino group, the PKK is eight. And so a pH of 3.6 is less than the PK of eight. And when the PK is greater than the pH, that means that it's gonna have the conjugate acid is gonna predominate. And the conjugate acid of the amino group is an NH three plus with a positive charge. So up here what we're gonna do for our amino group is we're gonna put a plus for the positive charge to consider the charge. So now let's move on to a Spartak acid. Since that's next in our chain here, a Spartak acids are group, and so we compare the pH to a Spartak acids are group, which is 3.9 and a pH of 3.6 is less than the ph of 3.9. So that means that the conjugate acid form of Spartak acids are group is gonna predominate and a Spartak acids can't get acid. It actually has a neutral charge, so it has a charge of zero. So in here, what we can do is just put zero because it has a neutral net charge at Ph. 3.6. So then we can check glue. Tannic acids are group, which is here, since it's next in our chain and blue tannic acids are group is here, it's P. K s 4.1, and so a ph of 3.6 is less than the P h. P. K A of 4.1. When the pH is less than the PKK. The conjugate acid predominates. The kanji acid of glue tannic acid, has a neutral net charge of zero. So we'll just put a zero in here. And then we take a look at the Car box, a group which is last, and the car boxing group, As you can see, which is way over here. It's PK is 3.5, and the PKF 3.5 is less than the pH of 3.6. And when the pH is greater than the P K A, the conjugate base predominates, and the conjugate base of the car box group is gonna be a car box late, and I on oops, with a negative charge, we'll put this in blue. And so all we need to do is total the net charges of each of these invisible groups to get the net charge of the structure at Ph. 3.6. So we have, ah, positive and a negative. So this group over here cancels out with this group over here, and then the other two groups are neutral. So that means that at this pH range at any pH in here are structure has a neutral net charge of zero and so recall that really, When we're calculating the P I, we want to find the pH range that has a neutral net charge of zero. So we were able to find it on our first try. Now, if we were to choose a different range if we had selected a different range over here, remember that when you cross the peak A that the net charge of the predominant structure changes by just one unit. And when you go from left to right, it goes down one charge. And so, from going from this yellow range over to this green range green range and crossing this PK here, we know that the predominant structure of the net charge gonna change. So any ph in this range, it's gonna go down by one. So we know it would be negative one over here. And so if we had chosen this pH range, then all we would have done is done the same cheat sheet. But we would have gone the opposite direction over here because we know that the charge would go up by one, and that would give us a net charge of zero. So it doesn't really matter which pH range that you choose. You choose one, determine the net charge and then you can use our cici ear to determine the net charge and a different range. So now that we found our neutral net charge, we have toe average the two PK sandwiching the neutral net charge. So the two PK sandwiching it are this one here and this one here so we can do R p I calculation over here. And it's just gonna be the average of these two numbers. So 3.9 plus 3.5 divided by two. And so this comes out to 7.4, divided by two, which comes out to 3.7. And what you'll see is that the p I of 3.7 matches our answer option A. So that means that a here is correct. We could give it a check, indicate that a here is correct and all our other ones are incorrect so we can cross them off. So this concludes our example here in our lesson, and we'll get some practice calculating the is electric point of peptides in our practice. So I'll see you guys in those videos

2

Problem

Estimate the isoelectric point of the following dipeptide in the figure.

A

4.75

B

7

C

9.5

D

10.5

3

Problem

Calculate the approximate pI of the peptide: C-G-E-K.

A

3.08

B

5.71

C

6.05

D

9.63

4

Problem

Draw in the R-groups for the following peptide & calculate the pI: ATLDAG.

A

7.41

B

6.9

C

9.25

D

3.7