Mark the approximate final position of the following tripeptid...

Question

Mark the approximate final position of the following tripeptide on the isoelectric focusing gel: Glu-Met-Asp.

Hint: calculate the isoelectric point of the peptide.

pH scale from 3 to 9, indicating the pH levels for isoelectric focusing. Table of ionizable groups with their pKa values for Glu, Asp, and N-terminus.

Accepted Answer

So this video is really gonna bring back a lot of great memories from our previous lesson videos, and that's because we're gonna need to remember how to calculate the isoelectric point of a peptide. And so if you don't remember how to calculate the isoelectric point of a peptide, make sure you go back to rewatch those lesson videos really quick before you continue here. And so this practice problem wants us to mark the approximate final position of the following tripeptide on an isoelectric focusing gel, and the tripeptide is glutamic acid, methionine, and aspartic acid. And so recall from our previous lesson video that isoelectric focusing is an electrophoretic technique that separates proteins based on their isoelectric points. And so the hint tells us to calculate the isoelectric point of the peptide. And notice that we have an isoelectric focusing gel provided over here on the right. And so, we need to mark the position that this peptide is going to, end up in after we run isoelectric focusing. Okay. So first, let's calculate the isoelectric point of this peptide. And so recall from our previous lesson videos that the first step in calculating the isoelectric point of a peptide is just to know how the ionizable groups of the amino acids ionize. And so, what that means is we need to know that glutamic acid has a negatively charged r group, and aspartic acid also has a negatively charged r group when it's ionized. And so, what we're gonna do is we're gonna redraw our same peptide, glutamic acid, methionine, and aspartic acid over here, and we're gonna consider all 4 of its ionizable groups. So we know that the n terminal end of every peptide has a potentially ionizable amino group. So let's put a little blank here for the charge of this amino group. And then we know that glutamic acid has a potentially ionizable charge. And the same goes for aspartic acid's R group, could potentially have a negative charge, and, the C terminal end of every peptide could potentially have a, negatively charged carboxylate anion. And so we'll leave these blank here for that. So notice that we're given this table over here with the, pKas of the amino acid residues, and we're given all of the appropriate pKas for the 4 ionizable groups that we need. And so the second step in calculating the isoelectric point of a peptide is to order all of the pKas from smallest to greatest. And so let's go ahead and do that here. So our smallest pKa is 3.5, and then, so that's our smallest one for the c terminus, which is our carboxyl group, so let's put carboxyl above it. Then our next smallest is 3.9, and that is for our aspartic acid r group. So we can put a s p for aspartic acid's r group. Then the next smallest is going to be 4.1, which is glutamic acid's r group, so we can put 4.1 and glutamic acid's r group. So we can put 4.1 and glutamic acid above it. And then the greatest pKa is gonna be pKA of 8, which is the pKA of the n terminal or, the pKA of the amino group, so we can put 8.0 and put amino above it. So now we've completed the second step in calculating the isoelectric point of a peptide. And the third step is to just, recognize that we're going to need to check any pH between the neighboring pKas and, determine the predominant structure and the net charge of the predominant structure in that region. So we can pick any region that we want between any of these pKas, but let's start and pick a a a region, a pH within, the region that's on the left since that is typically how we do things from left to right. So we can pick any pH that we want as long as it falls between these 2 pK as. So let's pick a pH of, let's say, 3.6. Why not? So we pick a pH of 3.6. And so, what you'll what we'll need to do is compare the pH that we've chosen to each of the individual pka as that are present. And so, let's start off by comparing the pH of the solution to the amino group pKa. And so notice that the pH of the solution, 3.6, is smaller than the pKa of the amino group. And recall from our previous lessons that when the pH is smaller than the pKa, that means that the conjugate acid predominates and the conjugate acid form of the amino group is gonna have a positive charge. It's gonna be an NH 3 plus. So for the amino group up here, let's just put positive charge on it so that we know that it's gonna be positively charged. So now let's move on to our next group, and our next group is glutamic acid. And so let's compare the pH to glutamic acid's, so let's compare the pH to glutamic acid's, pKa. And so the pH of 3.6 is smaller than the pKa. And again, when the pH is smaller than the pKa, the conjugate acid predominates, and the conjugate acid form of glutamic acid to r group is actually gonna have a neutral charge. And so we can go ahead and put a, 0 in here for its r group since it's gonna have a neutral charge. And, we can move on to our next group. So the pH of 3.6 is again smaller than the pKa of the aspartic acid r group. And so, again, when the pH is smaller than the pKa, the conjugate acid predominates, and the conjugate acid form of aspartic acid's r group has a neutral net charge of 0. And then for our final group, notice that the pH of 3.6 is actually greater than the p k a of the carboxyl group. And so when the pH is greater than the pKa, the conjugate base predominates. And so the conjugate base of the carboxyl group is actually going to have a negative charge, so let's put a negative charge over here. So what we're essentially saying is that within this yellow region, within any pH in this yellow region, the predominant structure of our peptide is going to have a neutral net charge since, the positive charge on the amino group cancels out with the negative charge on the carboxyl group, and, none of the r groups have a charge. So our peptide has a neutral net charge of 0. And when we're considering and looking for the isoelectric point, that's exactly what we're looking for, is the region that has, the predominant structure with a neutral net charge. And so what that means is all we need to do to get the p I is to take the average of the 2 pKas that sandwich the region that contains the neutral net charge. And so those 2 pKas are the pKa of the carboxyl group and the pKa of aspartic acids, r group. And so let's go ahead and calculate for the p I. So the p I or the isoelectric point is gonna be the average of those 2 pKas, so 3.5 plus 3.9 divided by 2, and so this comes out to 7.4 divided by 2, and if you type in your calculator 7.4 divided by 2, you get an answer of 3.7. And so what that's saying is that the p I or the isoelectric point of our peptide, glutamic acid, methionine, aspartic acid, is 3.7. And so, we know that, the peptide is going to continuously travel through the isoelectric focusing gel until it reaches the position on, the gel where the pH is equal to the p I. And so, what we essentially want to do is we want to mark the position of our peptide on this gel. And so we know that our peptide is going to travel to the pH that matches the p I, so we wanna, mark the pH of 3.7. And so here we have a pH of 3, here we have a pH of 4, and so a pH of 3.7 would be somewhere in this region right below the pH of 4. And so this is the position where the pI is, the isoelectric point, and this position, marked in red, is the final position where we expect our peptide to, end up in, after isoelectric focusing is complete. And so this concludes our practice problem here, and I'll see you guys in our next video.

Biochemistry 때문에 힘들어하고 계신가요?

Mark the approximate final position of the following tripeptide on the isoelectric focusing gel: Glu-Met-Asp.
Hint: calculate the isoelectric point of the peptide.

다음 시청

Isoelectric Focusing 연습 세트

Biochemistry 때문에 힘들어하고 계신가요?

Mark the approximate final position of the following tripeptide on the isoelectric focusing gel: Glu-Met-Asp.Hint: calculate the isoelectric point of the peptide.

다음 시청

Isoelectric Focusing 연습 세트

Mark the approximate final position of the following tripeptide on the isoelectric focusing gel: Glu-Met-Asp.
Hint: calculate the isoelectric point of the peptide.