Titrations of Amino Acids with Ionizable R-Groups - Online Tutor, Practice Problems & Exam Prep

Amino acids with ionizable R groups possess three ionizable groups: the amino group, carboxyl group, and R group, leading to three inflection points in their titration curves. For example, histidine's titration curve reveals pKa values for its carboxyl group, R group, and amino group, indicating their protonation states at various pH levels. The isoelectric point (pI) is calculated as the average of the pKa values surrounding the neutral net charge, demonstrating the relationship between pH and charge in amino acids. Understanding these concepts is crucial for amino acid analysis and protein structure determination.

Titrations Of Amino Acids With Ionizable R-Groups

In this video, we're going to talk about the titrations of amino acids with ionizable R groups. So, recall that amino acids with ionizable R groups actually have 3 ionizable groups: their amino group, their carboxyl group, and their R group. Also, recall that there are 7 amino acids with ionizable R groups, and our mnemonic to remember those 7 amino acids is "just yucky crazy dragons eat knights riding horses." If you can remember this mnemonic, you'll remember those 7 amino acids with ionizable R groups. Because they have 3 ionizable groups, that also means that they're going to have 3 inflection points and equivalence points on their titration curves. And really, that's the biggest difference between the titrations of amino acids with ionizable R groups and titrations of amino acids with non-ionizable R groups. Remember, amino acids with non-ionizable R groups only have 2 ionizable groups, so they only have 2 inflection points and equivalence points in their titration curves. But again, ionizable R groups are going to have 3, not 2 inflection points and equivalence points.

In our example, what we're going to do is review some titration curves, but specifically for an amino acid with an ionizable R group. Here, we've got the titration curve of histidine, which is indeed one of our 7 amino acids with an ionizable R group because histidine's one-letter code is H. If we look at our example, you'll see we've got these boxes all around the titration curve, asking us questions about the titrations, and they're color-coordinated to specific regions on the titration curve. We're going to start with this black box in the bottom right. It's asking us what the black curve represents. This black curve refers to histidine's titration curve.

The next box, this green box in the bottom left, asks us what the green dot and lines represent. The green line, this green dot, and this green line indicate where the curvature of histidine's titration curve is changing. When the curve changes in that fashion, it corresponds with a midpoint. We know midpoints are associated with pKa values. Here, we see a pKa value of about 1.82, which we can associate with the carboxyl group pKa because carboxyl groups have pKas in the ballpark range of about 2.

The next box is this blue box, and it's asking us what the blue dot and lines represent. Here, the curve changes its curvature before and after this blue dot, indicating another midpoint. This is the pKa of the R group. The orange box asks what the orange dot and lines represent. Again, the curvature changes here, indicating a midpoint and pKa at 9.17, which we know is associated with the pKa of the amino group, since their pKas fall in the range of about 9 to 10.5.

This is the biggest difference between the titration curves of amino acids with ionizable R groups because they actually have 3 midpoints, whereas with our previous curves with amino acids with non-ionizable R groups, there were only 2 midpoints. The next question concerns the pink line, which shows up at 1 molar equivalent of the titrant being added, indicating the equivalence point of the carboxyl group. At this equivalence point, all following instances will only show the deprotonated form of the carboxyl group.

The yellow line represents another equivalence point, this time for the R group, as indicated by its position before the pKa of the amino group and after the pKa of the r group. The last box discusses the light blue line, which corresponds with 3 molar equivalents of titrant being added, indicating the final equivalence point for the amino group. Once this point is reached, all remaining titration will show the deprotonated form of the amino group.

In our next video, we're going to discuss how to draw amino acids with ionizable R groups just by looking at their titration curves. Again, the biggest difference here is that there are 3 midpoints and 3 equivalence points, whereas previously we only had 2 midpoints and 2 equivalence points. That concludes this lesson; I'll see you guys in our next video.

Titrations Of Amino Acids With Ionizable R-Groups

So in our last lesson video, we reviewed the titrations of amino acids with ionizable R groups. And in this video, we're going to talk about how to draw the predominant structures of amino acids with ionizable R groups straight from their titration curves, and we'll also review how to calculate their isoelectric points.

And so the isoelectric point (pI) is always going to be equal to an equivalence point, but it will not always be equal to the carboxyl group equivalence point, like it was with amino acids with non-ionizable R groups. Nevertheless, it's still equal to an equivalence point, and it will always be equal to the equivalence point that allows for the neutralization of the net charge, meaning that there's going to be a net charge of 0 at the isoelectric point or the pI.

Now, in our example below, we're first going to draw the predominant structures of histidine at each of its colored regions on its titration curve. And then, we'll go back and calculate its pI. Notice down below in the center, we have the titration curve for histidine, and it's colored into these different regions. We're going to start off here with the red region and draw the predominant structure of histidine in this red region, in this red box over here on the left.

Notice that this red region here corresponds with pH values that are less than about 2, and these pH values that it corresponds with are less than the pKas for its 3 ionizable groups. And that's exactly what's being indicated over here on the left, that the pH is less than the value of all of the other pKas and pKRs. Thus, when the pH is less than the pKa, that means that the conjugate acids are going to predominate, and the conjugate acids are all protonated. Let's go ahead and draw histidine's structure with protonated groups. So the amino group is going to be protonated, so it'll be NH₃⁺. The carboxyl group is also going to be protonated, so it'll be OH neutral. And then its R group, recall, is literally just alanine, so CH₂, with a 5-membered ring branching off, so it'll be a 5-membered ring, like that. Recall that histidine's R group is comparable to a horse, so it's got these back legs, these front legs, and then 2 nitrogens, one of them is at the neck and the other one is not being stepped on by the horse. Remember that it's the nitrogen that's at the neck that's going to be the one that's ionizable, so the one that's going to have the positive charge on it.

All three of these groups plus 2 is going to be plus 2 in this red region. Now moving on to the next region, what we have is the yellow region, which corresponds with pH values between, 6 and 2. These pH values that correspond with the yellow region are, still lower than the pKa and the pKr of the amino group, but now greater than the pKa of the carboxyl group. So only the carboxyl group is going to change in this yellow region down below. And that's exactly what's being indicated here, that the pH is now greater than the pKa of the carboxyl group. Essentially, what we can do is copy the same exact structure that we had drawn earlier and bring it down here. Essentially, the only thing that's going to change is that the carboxyl group is now going to be deprotonated and in the conjugate base form since the pH is greater than its pKa. Notice that the total net charge here, we have 2 positives and one negative, that means that the total net charge is going to be plus 1.

Now moving on to the next region, which is the light blue region, it corresponds with pH values that are between 9 and about 6, and these pHs are now greater than both the pKa and the pKr of the carboxyl group. That's exactly what's being indicated here, and all we need to do is draw the same exact structure that we had over here on the left, and just change the pKr, since that's the only one that's being changed from this one that's on the left to this one over here that's on the right. When we change this, what you'll see is that we have 2 charges, and they cancel each other out, this positive and this negative charge.

That means that we have a neutral net charge of 0 on this blue structure. And then, of course, in the pink structure, it corresponds with pH values that are above 9, and these pH values are greater than all of the other pKas. And that's exactly what's being indicated here. So when the pH is greater than the pKa, the conjugate bases predominate, and it's going to be exactly the same structure that we drew down below, except, the amino group is now finally going to change. We're now going to have a deprotonated conjugate base form of the amino group, and now the total net charge is just going to be negative one.

Essentially, what you'll see here is that this red region here has a net charge of plus 2, indicated by this over here. The yellow region in here has a net charge of plus 1, indicated down below here. In the blue region, we said that there was a net charge of 0, and in the pink region, there is a net charge of minus 1. It's important to note here that the net charges of the predominant structures decrease. Notice that they're going down, they're going from plus 2 down to negative 1, as the pH is increasing. Also, notice that the net charges go down by one as we cross a pKa. It goes from plus 2 in the red region, and once we cross this green pKa into the yellow region, it goes down one charge. The same goes for each of these other pKa's - when you cross a pKa, it goes down one charge as you go from left to right.

And now we're going to calculate the isoelectric point, which we know is going to, again, be at the equivalence point that allows for neutralization of the net charge. Again, the region that has a neutral net charge is the light blue region. We're going to essentially take the average of the 2 pKa's sandwiching the region that has a neutral net charge on the predominant structure. So, it's these 2 pKa's, PKa+9.17÷2. This comes out to about 7.59. This is going to be the isoelectric point of histidine. Up above here, we can indicate that 7.59 is the isoelectric point, and you can see that it's showing up right at the equivalence point here of the R group, shown over here.

Essentially, this concludes our lesson on how to draw the amino acids of ionizable R groups from their titration curves, and we'll be able to get some practice utilizing these techniques here in our next practice video. I'll see you guys there.

Determine the pI of Glu & mark it on the provided titration curve.

a. 6.96                                          

b. 5.93

c. 7.48

d. 3.22

Use the titration curve to draw the predominate structures of Glu at pH = 1.0, pH = 3.0, pH = 7.0, & pH = 11.0.

Glu at pH = 1.0                  Glu at pH = 3.0                  Glu at pH = 7.0                   Glu at pH = 11.0

Alright. So here's a 2-part practice problem broken up into part A above and part B below. Part A wants us to determine the PI or the isoelectric point of glutamic acid, and then to mark it on the provided titration curve on the right. We know from our previous lesson videos that the first step to determine the isoelectric point is to understand how the R group ionizes. We can remember our mnemonic for the 7 amino acids with ionizable R groups, which is "yucky crazy dragons eat knights riding horses." Glutamic acid's one-letter amino acid code is an E, so it is part of our mnemonic. Because dragons eating things is a negative thing, it reminds us that glutamic acid has a negatively charged R group. The second step in determining the isoelectric point is to order the pKa values from the smallest to the largest value; note that the pKas are already ordered from smallest to largest in our titration curve. The alpha carboxyl groups have pKa's in the ballpark range of about 2, so the first pKa corresponds to the carboxyl group. Alpha amino groups typically have pKa's around 9 to 10.5, so the third pKa corresponds to the amino group. The pKr is the pKa of the R group. Our third step is to use the middle pKa as a guide and determine the net charges of the predominant structures at any pH between the adjacent pKas. This will align with the regions indicated between the adjacent pKas on the titration curve. Starting with the region to the left, we pick a pH of 3 and compare it to all of the pKa values. The pKa of the amino group, which is 9.67, is greater than the pH. Therefore, the conjugate acid predominates, resulting in a protonated amino group (NH₃⁺). The pKa of the carboxyl group is smaller than the pH of the solution. Thus, the conjugate base predominates, meaning the carboxyl group is deprotonated and negatively charged. For the R group of glutamic acid, since pKr is 4.25 and greater than the pH, the conjugate acid predominates in the neutral protonated form (OH). With one positive and one negative charge cancelling each other out, the predominant structure has a net charge of 0. Therefore, to find the pI, average the two pKas (carboxyl and pKr) surrounding the region where the structure has a neutral net charge, calculating to about 3.22. This value matches option D, indicating a correct answer for part A. Mark this pI on the titration curve slightly above 3 on the pH scale, corresponding to this position in the yellow region. Moving to part B, we need to use the titration curve to draw the predominant structures of glutamic acid at the indicated pH values. Starting at pH 1, this is below the pKa values for all ionizable groups, and the conjugate acid form predominates, showing an NH₃⁺ for the amino group and OH for the carboxyl groups, cumulating in a net charge of +1. At pH 3, near the isoelectric point, the structure maintains a neutral charge, with only the carboxyl group deprotonating to reflect the conjugate base form. At pH 7, transitioning through the pKr, adjusts the net charge to -1, now changing only the R group while maintaining the rest. Finally, at pH 11, above all pKas, all ionizable groups are in their conjugate base form, resulting in a net negative charge of -2. This concludes the explanation for this scenario, looking forward to engaging in our next video.

Draw the predominate structures of K at the indicated sections of its titration curve. Mark the pI on the curve.

So this practice problem wants us to draw the predominant structures of lysine at the indicated sections of its titration curve. And then it also wants us to mark the position of the pI or the isoelectric point on the curve. Looking at the titration curve shown below, notice that it's indicating the positions of the pKas for lysine's 3 ionizable groups. It's also not giving us the exact values of these pKas, but we actually do not need the exact values to solve this problem. Looking at the first pKa, pKa1, notice that it corresponds with the position on the curve right here, which correlates with pH values right around 2. Alpha groups typically have pKas in the ballpark range of about 2, indicating that this pKa must be the alpha carboxyl group pKa. I've labeled it accordingly. For similar reasoning, this pKa up here must be the alpha amino group pKa, because it corresponds with pH values right around 10, and alpha amino groups have pKas in the range of about 9 to about 10.5. The pKr is the pKa of the r group, and I have labeled it as such. Now, we can take a look at the first region, the red region, which corresponds with the low pH values shown below all of the pKas. Recall that when the pH is lower than the pKa, the conjugate acid form or the protonated form exists. In the red region, our predominant structure will have the protonated form for all 3 of its ionizable groups. We'll have an NH3+ for the amino group. For the carboxyl group, we'll have an OH, which will be protonated and will be neutral. The R group has a four-carbon chain starting and an amino group at the very end. This amino group will also be in the conjugate acid protonated form, so it will be an NH3+. Here, we have 2 charges on our predominant structure, both positive, providing an overall charge of +2 in this red region. Moving to the next region, the blue region, it overlaps with the curve between these 2 pKas, indicating pH values right in between. Here, the pH is still lower than the pKas of the amino group and the R group, so those will remain unchanged. However, it's now greater than the pKa of the carboxyl group. Therefore, the only change from this structure to the blue region is the carboxyl group, which will now be in the deprotonated form, as a carboxylate anion. The NH3+ in the R group will remain the same, and the total net charge is now +1. In the yellow region, the pH is now greater than the pKa of the amino group but still lower than the pKa of the R group. The only change here is the amino group, which will transition to a deprotonated NH2 form, now neutral. The carboxyl group will remain as a carboxylate anion, and the amino group in the R group will still be an NH3+. The charges offset, resulting in a neutral net charge of 0, which is the predominant structure in the yellow region. Finally, for the purple region with pH values much higher than any of the pKas, all ionizable groups will be in their deprotonated forms. The amino group will be NH2, neutral. The carboxyl group will be a carboxylate anion, carrying a negative charge. The R group will also be NH2, neutral. With only one negative charge overall, the net charge is -1. Notably, as the pH increases, the charges of our predominant structures decrease. The pI, the exact pH where the predominant structure has a neutral net charge, is found in the yellow region. The isoelectric point lies at the average of the two pKas surrounding the region where the net charge is neutral, marking it as the pI. This concludes the discussion on this practice problem.

Label each arrow on Asp's titration curve & draw its predominate structure at each colored region.

A) Amino group pK_a.

B) Carboxyl group pK_a.

C) R-group pK_a (pK_a = 3.9).

D) Amino group equivalence point.

E) Carboxyl group equivalence point.

F) R-group equivalence point.

Alright. So this practice problem wants us to label each arrow on aspartic acid's titration curve and then to draw aspartic acid's predominant structure at each of the colored regions on the titration curve. We'll start by labeling each arrow, and the first arrow here points right at this position, which corresponds with half of the molar equivalents of titrant being added. This indicates it's some kind of midpoint or inflection point, and every midpoint or inflection point corresponds with a pKa value. As it corresponds with a pH of about 2 and we know that alpha carboxyl groups have pKas in the ballpark range of about 2, this must be the pKa of a carboxyl group, which matches up with answer option B. So, we label b on this first arrow and cross off B from our list. The next arrow points at a position corresponding with exactly 1 molar equivalent of titrant being added, signaling an equivalence point since this is the end of an acid being completely neutralized. This must be the equivalence point of the carboxyl group, labeled as E in answer option E. The subsequent position, occurring at 1.5 molar equivalents, indicates another midpoint, with half of another acid being neutralized. This point correlates with a pH just under 4. Option C indicates the R group has a pKa just below 4, at 3.9, hence this must be the midpoint and the pKa for the R group. We label C here. The next point appears at 2 molar equivalents of titrant being added, denoting we've neutralized another acid. Since this is an equivalence point and the only pKa that comes before it that has not already been neutralized is the alpha-amino group, we label this position A. Another midpoint is observed at 2.5 molar equivalents, indicating another acid being half neutralized. This pKa shows up around 9. Alpha carboxyl groups have pKas in the range of about 9 to 10.5, suggesting this must be the pKa of an amino group. We label this A and cross off A from the list. The final point, corresponding with exactly 3 molar equivalents of titrant being added, is the amino group equivalence point, labeled D. Now that we've labeled each arrow on aspartic acid's titration curve, we'll draw aspartic acid's predominant structure at each colored region. In the green region, the pH values are lower than all of the pKas, causing the conjugate acid forms to predominate for all the ionizable groups. The amino group is protonated as NH3+, the carboxyl group as COOH, and the R group of aspartic acid also protonated. This gives our structure a net charge of +1 in the green region. In the red region, the only change is with the carboxyl group transitioning to its conjugate base form, COO-, while all other pKas are greater than this pH, remaining the same. This results in a net charge of 0 for this region. The blue region includes pH values greater than both the pKa of the R group and the carboxyl group. The difference in this structure is the R group converting to its deprotonated conjugate base form, and the carboxyl group remains the same, resulting in two negative charges and one positive charge. The net charge here is -1. The last region, the pink region, corresponds with very high pH values, exceeding all pKas. All ionizable groups are in their conjugate base forms: the amino group as NH2, the carboxyl as COO-, and the R group also as a negative charge. This establishes a net charge of -2 for this region. This practice problem has demonstrated the step-by-step process of identifying key points in a titration curve and relating them to the charged states of aspartic acid at differing pH levels. I'll see you guys in our next video.

Here’s what students ask on this topic:

What are the seven amino acids with ionizable R groups?

The seven amino acids with ionizable R groups are Tyrosine (Y), Cysteine (C), Aspartic acid (D), Glutamic acid (E), Lysine (K), Arginine (R), and Histidine (H). A mnemonic to remember these is 'Yucky Crazy Dragons Eat Knights Riding Horses'. These amino acids have three ionizable groups: the amino group, carboxyl group, and the R group, which influence their titration curves and pKa values.

How do you determine the isoelectric point (pI) of an amino acid with an ionizable R group?

The isoelectric point (pI) of an amino acid with an ionizable R group is determined by averaging the pKa values that surround the region where the amino acid has a net charge of zero. For example, for histidine, the pI is calculated as the average of the pKa values of the carboxyl group and the R group, which are 6.0 and 9.17, respectively. The formula is:

$\frac{6.0+9.17}{2}$

This results in a pI of approximately 7.59.

What is the significance of the inflection points on the titration curve of an amino acid with an ionizable R group?

The inflection points on the titration curve of an amino acid with an ionizable R group correspond to the pKa values of its ionizable groups. These points indicate where the amino acid undergoes protonation or deprotonation. For example, histidine has three inflection points corresponding to the pKa values of its carboxyl group, R group, and amino group. These points are crucial for understanding the amino acid's behavior at different pH levels and for calculating its isoelectric point (pI).

How do you draw the predominant structure of an amino acid at different pH levels using its titration curve?

To draw the predominant structure of an amino acid at different pH levels using its titration curve, identify the pH range and compare it to the pKa values of the amino acid's ionizable groups. If the pH is less than a group's pKa, the group will be protonated. If the pH is greater, the group will be deprotonated. For example, at pH < 2, histidine's amino, carboxyl, and R groups are protonated. At pH 2-6, the carboxyl group is deprotonated. At pH 6-9, the R group is deprotonated. At pH > 9, the amino group is deprotonated.

What is the difference between the titration curves of amino acids with ionizable R groups and those without?

The main difference between the titration curves of amino acids with ionizable R groups and those without is the number of inflection and equivalence points. Amino acids with ionizable R groups have three ionizable groups (amino, carboxyl, and R group), resulting in three inflection points and three equivalence points on their titration curves. In contrast, amino acids without ionizable R groups have only two ionizable groups (amino and carboxyl), leading to two inflection points and two equivalence points.