Isoelectric Point - Online Tutor, Practice Problems & Exam Prep

The isoelectric point (pI) of an amino acid is the pH at which it has zero net charge, maximizing zwitterion concentration. For generic amino acids, pI is calculated by averaging the pKa values of the carboxyl and ammonium groups, typically yielding a pI around 5.5. Amino acids are categorized into three groups for pI calculation: non-acidic/basic, acidic/basic, and cysteine. Cysteine's pI involves averaging its unique pKa values, while acidic and basic amino acids require selecting the two most similar pKa values for accurate calculation.

Definition of Isoelectric Point

In this video, we're going to discuss a really important property of amino acids called the isoelectric point. So the isoelectric point is the pH at which an amino acid has zero net charge. It's also defined as the pH at which an amino acid has the greatest concentration of zwitterions present, which is what I have here. Now, those two definitions actually mean the same exact thing because think about it. What type of net charge does a zwitterion have? Zero. Remember, by definition, zwitterions have zero net charge, right? So if I have the maximum concentration of zwitterions, what is my net charge going to be? Zero, okay? So, it's two different ways of saying the same thing which is that we're looking, we're kind of tuning it. We're going to tune that pH to figure out what's the pH that I can set it at for that amino acid so that I have the most amount of zwitterions present aka so I have the least amount of net charges present, okay, so that they're all at zero.

So for a generic amino acid, the pI would just be calculated by averaging the pKa of the two functional groups. Now, what do I mean by R? It's unknown. I don't know what it is. So let's go ahead and do this exercise first. Before we talk about specific amino acids, let's try to approximate what the general pI would be for just a generic amino acid. So the way that this would work is that we would just have to use approximate pKa values.

So what did I tell you guys is the approximate pKa value for the O⁻ on an amino acid? It's around 2, right? So let's go ahead and fill that in here. It's around 2 in the pink area. Now, for the blue area, the basic area, what's the approximate pKa for an amine on an amino acid? For actually, it's called the ammonium group because it has a positive charge. It's around 9. Remember that?

So for just some random amino acid where you don't know what the R group is, the isoelectric point would be taken by just averaging the pKa's of both of these groups. Now, this number would come out to 112, which if you type it into your calculator, that's going to give you an isoelectric point of 5.5, okay? And it turns out that that actually is close to many of the answers. For lots of the amino acids, our isoelectric point will be around 5.5. That means that if you set your pH to 5.5 and put these amino acids in, most of them are going to be zwitterions because it's going to be in between the two pH's of your pKa1 or in between the pKa1 and pKa2. It's going to be at that midpoint so that most of them are zwitterions, okay? Now, it turns out that some of them are not going to be 5.5 but this is where most of them are going to be around, okay?

So how do we calculate the exact isoelectric points? We're going to discuss that in the next video.

Isoelectric Point of Non-Charged Amino Acids

So all 20 amino acids fall into 3 different buckets of ways to calculate the isoelectric point. I just want to look at what those categories are first. The first category is non-acidic or basic amino acids. That's group 1. Group 2 is acidic and basic amino acids. And group 3 is just cysteine. Cysteine is its own exception so it's going to be in its own bucket. So what that means is that if we were to work backwards from this, the isoelectric point of cysteine is in its own group. The ones that are in the acidic and basic amino acids group, what are they? Well, we should have that memorized. We should know that the acidic ones are ED. And we should know that the basic ones are KHR, right? So that means that if my amino acid is in that bucket, I use this technique. So that means that what types of amino acids are in the first bucket, almost all of them. So I'm just going to put here everything or I'm just going to put here all minus E, D, K, H, R, and C. Okay? So all of them, all 14 of them besides those will be in this first bucket. So let's talk about that first category. So if you have one of the amino acids that's not in these special categories, you can just calculate it as a generic amino acid which is the best part. All you do is you just look up the pKa values and average them. That means 14 of 20 are really straightforward. So, is phenylalanine in this first group? Yes. Because it's not acidic. It's not basic. It's not cysteine. So what I do is I have to look up the exact values for phenylalanine. Let's go ahead and do that now. What I see is that my values are 1.83 and 9.13. So let's go ahead and write those down. So it was 1.83 and, nope, 9.13. I almost got that wrong. Okay. Dyslexia, you did not win. So basically, those are my two values. So here, we're just going to have to use a calculator and average them. What that means is that I'm going to add 1.83+9.13/2 and whatever that's equal to will be the answer. So I'm just going to calculate it right now on my phone. So 1.83 + 9.13 equals 10.96 divided by 2 equals that the isoelectric point of phenylalanine is 5.48. Cool? Awesome. So let's go ahead and do the next one.

Isoelectric Point of Charged Amino Acids

In this next category, we have acidic and basic side chains. And these are going to have to use a different rule. And the reason is because you can't just average them. There are now 3 ionizable groups. And when you have 3, you don't just average 3 of them. You are still only supposed to use 2. So how do you pick the 2 that you're supposed to use? Well, what you do is you pick the 2 similar groups. You look for the 2 most similar groups, and then those are the \(pK_a's\) that you use. Now, I already drew out the 3 \(pK_a's\) for you that I got from the sheet, but just be aware that we're only going to use 2 of these. 1 of these is going to get scratched out. It's not going to get used for the isoelectric point. So the place that you always start is with the side chain because the side chain is always going to be one of your 2. So let's circle a side chain, highlight it. For sure, I'm going to use this \(pK_a\). That \(pK_a\) is 6. So for sure, that's going to be one of them. But now, I have to decide, is the nitrogen group more similar or is the oxygen group more similar to the histidine side chain? And the answer is the nitrogen, okay? Why? Guys, it just has to do with atoms. It's that easy. If you have nitrogens in your side chain, you pick the ammonium to be the most similar. If you have oxygens in your side chain, you pick the oxygen to be most similar. So if this had been glutamic acid that has carboxylic acids on both sides, you pick the \(O'\)s. But if this was arginine which nitrogens on one side, then you'd pick the nitrogen and the nitrogen is being similar, okay? In this case, histidine has what? Nitrogens. So I would pick the nitrogen group. What that means is that I'm going to use this as my second group. So I'm going to use this \(pK_a\) as my second \(pK_a\). And I'm scratching out \(pK_a\) 1 because I'm not going to use it. That's not the most similar. And now, we average these out. So this part's easy, 6.00+9.172. So we're just going to use our calculator again. 6 + 9.17 equals 15.17 divided by 2 equals that the isoelectric point of histidine is 7.59. I'm rounding up. Okay? Awesome. So we're done with this one. Let's move on to the next category.

Isoelectric Point of Cysteine

So technically, cysteine doesn't need to have its own category. But I put it here because I realized that it could be the most confusing one. And the reason is that cysteine is the only ionizable side chain that has neither an oxygen nor a nitrogen, right? It's the only one that has a heteroatom that's different. So, you could be really confused trying to pick which one's similar. It's the same rule, but you might just have a hard time applying it. You might say, well, is the sulfur more like the nitrogen or is it more like the oxygen? I just wanted to make it clear to you guys that for the purpose of this exercise, it is more similar to the oxygen. And it makes sense because remember the oxygen is either going to have a hydrogen or a negative charge, right? Same thing with the sulfur. It's either going to have a hydrogen or a negative charge. It's never going to have 2 Hs and a positive. So this is just basically an extra little point to say that you average the sulfur and the oxygen as the ones being similar. It's basically the same thing as the acid and base rule. It's just a different application of it. Okay?

So that means that the pK_as that I'm using here well, I'm going to use these two groups which means the pK_as I'm using are pK_a 1 and pK_a 3 and I'm not using pK_a 2. So now we're just going to add them together. So it's going to be 1.96+8.18 /2. So I'm going to do that in my calculator. So I'm getting 10.142 equals an isoelectric point of 5.07. Cool? Awesome, guys. So we're done with this page. Let's move on to the next.

Calculate the isoelectric point of tyrosine (Y)

Calculate the isoelectric point of tyrosine. Great. So remember that all we need for this are the pKas of the molecule, and then we need to know which pKas to use. Remember, this has nothing to do with an initial pH. We just have to average out the numbers. Tyrosine just because, like, we should always draw it out even though it didn't actually ask me to. But it's just always a good idea to practice that. Okay? So there we have tyrosine. And, well, actually, we need to do that so we could pick which pKa, or yeah, which pKas to use. So we know that this is not acidic and it's not basic. So what do I have to do? This is not in my acidic category. What category is it? Is it acid–base? No. Is it cysteine? No. So that means that this would just go into my typical bucket of average the 2 pKas, pKa₁ and pKa₂. So that means I don't need to worry about pKa₃ because this is not an acid or a base, right? Is tyrosine in my it's in my polars but it's not an acid and base. Remember? So that means that I can just go ahead and average these out. So this is gonna be this one's number 2.20. This one's 9.11. And then I'm not using this one over here. So that's going to be 2.20 + 9.11 2 My isoelectric point is 5.65. And we're done. It was that easy. So realize that the first step was recognizing what category it was in. Remember that tyrosine was in the non-acidic, non-basic, non-cysteine category, so then it's really easy. We don't even worry about the side chain. We just look at this one. Okay? Great. So let's move on to the next.

Calculate the isoelectric point of glutamic acid (E)

Calculate the isoelectric point of glutamic acid. So let's go ahead and draw this guy out. So we have On. Remember that glutamic acid is a carboxylic acid with 2 carbons. So, I've already written out our pKas for us and we need to decide which 2 we are using. Okay? Remember that this is going to be pKa1, this is going to be pKa2 and this is going to be pKa3 over here. So we know for sure we are using the side chain. Because the thing is, glutamic acid is in our acid-base category. So since it's in our acid-base category, we need to use the side chain first. But then the question is which other value do we use? pKa1 or pKa2? Since I have an oxygen, and oxygen is most similar to oxygen, I'm going to use this one. Great. And what that means is that now I just use these values. I can scratch out pKa2. And this is going to be 2.19. This is going to be 4.25. So now I go ahead and add these up. And what this is going to give me is 2.19 + 4.25 = 6.44 ÷ 2 = 3.22. Okay? We're done with this question so let's move on.

Here’s what students ask on this topic:

What is the isoelectric point (pI) of an amino acid?

The isoelectric point (pI) of an amino acid is the pH at which the amino acid has zero net charge. At this pH, the amino acid exists predominantly as a zwitterion, meaning it has both positive and negative charges but the overall charge is neutral. For a generic amino acid, the pI can be calculated by averaging the pKa values of the carboxyl group (typically around 2) and the ammonium group (typically around 9). This usually results in a pI around 5.5. However, the exact pI can vary depending on the specific amino acid and its side chain properties.

How do you calculate the isoelectric point of a generic amino acid?

To calculate the isoelectric point (pI) of a generic amino acid, you average the pKa values of its carboxyl and ammonium groups. The carboxyl group typically has a pKa around 2, and the ammonium group has a pKa around 9. The formula for calculating the pI is:

$\frac{({\mathrm{pK}}_1+{\mathrm{pK}}_2)}{2}$

For example, if pKa1 is 2 and pKa2 is 9, the pI would be:

$\frac{(2+9)}{2}=5.5$

Why is cysteine considered a special case when calculating its isoelectric point?

Cysteine is considered a special case when calculating its isoelectric point (pI) because it has a unique ionizable side chain containing sulfur, unlike other amino acids that have oxygen or nitrogen in their side chains. This can make it confusing to determine which pKa values to use. For cysteine, you average the pKa values of the carboxyl group and the thiol group (sulfur-containing side chain), ignoring the ammonium group. This is because the thiol group is more similar to the carboxyl group in terms of ionization behavior. This special consideration ensures accurate pI calculation for cysteine.

How do you calculate the isoelectric point for amino acids with acidic or basic side chains?

For amino acids with acidic or basic side chains, you cannot simply average all three pKa values. Instead, you select the two most similar pKa values. First, identify the pKa of the side chain. Then, determine which of the remaining pKa values (carboxyl or ammonium) is more similar to the side chain pKa. For example, if the side chain contains nitrogen, you would choose the ammonium pKa. The formula for calculating the pI is:

$\frac{({\mathrm{pK}}_{\mathrm{side\; chain}}+{\mathrm{pK}}_{\mathrm{similar}})}{2}$

This method ensures accurate pI calculation for amino acids with ionizable side chains.

What are zwitterions and how do they relate to the isoelectric point?

Zwitterions are molecules that have both positive and negative charges but an overall neutral charge. In the context of amino acids, a zwitterion form occurs when the amino group is protonated (NH₃⁺) and the carboxyl group is deprotonated (COO^-). The isoelectric point (pI) is the pH at which the concentration of zwitterions is maximized, meaning the amino acid has zero net charge. At this pH, the amino acid does not migrate in an electric field, making the pI a crucial property in techniques like isoelectric focusing and protein purification.