Isoelectric Point - Online Tutor, Practice Problems & Exam Prep

The isoelectric point (pI) of amino acids is the specific pH at which a molecule has no net charge, balancing positive and negative charges. To calculate pI, average the two relevant pKa values, particularly for amino acids with non-ionizable R groups, which have only two pKa values. More acidic amino acids have lower pI values, while basic amino acids have higher pI values. At pH equal to pI, molecules do not migrate in an electric field, making pI crucial for techniques like electrophoresis, which separates proteins based on charge.

Isoelectric Point of Amino Acids

In this video, we're going to begin our discussion on the isoelectric point of amino acids. So the isoelectric point can actually be abbreviated with the letters pI, which is shown here in parentheses. And, really, the isoelectric point is just a pH value. It's a specific or an exact pH at which a molecule is going to have no net charge. If it has no net charge, it means that if there are any positive charges, they're going to be completely balanced by negative charges because the overall net charge of the protein, or of the amino acid, is going to be 0 when the pH is equal to the isoelectric point. In our practice videos for this topic, we're going to be asked to calculate the isoelectric point of amino acids. It's important to keep in mind that the isoelectric point is always just the midpoint or the average between just 2 pKa values. This is crucial because even if there are 3 pKa values given, or 4, or 5, or however many they give you, the isoelectric point is always just an average of just 2 pKas. Specifically, the way that you know which 2 pKas to average is going to have to do with the 2 ionizations that involve the neutral species. I want you guys to know that when you're calculating the pI in our practice videos later on, you really just want to ensure that you're only averaging 2 pKas. That's all I want you guys to know for now.

Another important trend to keep in mind is that more acidic amino acids have more acidic isoelectric points, and the same applies to basic amino acids; the more basic ones have more basic pI values. That's an important trend to keep in mind.

The last thing I want to leave you guys off with is that when the pH is equal to the pI of a molecule, we already know that the molecule is going to have no net charge, as already stated earlier. But what's also important to know is that the molecule, when it has no net charge, it's actually not going to migrate in an electric field. This is why the isoelectric point is such a valuable property because scientists can adjust the pH to try to get it to equal the exact pI of a molecule. This means that it's going to have no net charge, and they can use the isoelectric point of a molecule in a technique called electrophoresis. Electrophoresis basically allows scientists to separate proteins that have a net charge using an electric field. We'll talk a lot more about electrophoresis in another topic later on in our course in a different video. But for now, all I want you to know is that the pI, or the isoelectric point, of a protein or an amino acid is a useful property because it allows them to separate and isolate specific amino acids and proteins of interest using electrophoresis. In our next video, we're going to talk about how to calculate the isoelectric point of an amino acid with a non-ionizable R group. So, I'll see you guys in that video.

Isoelectric Point of Amino Acids

So in our last video, we said that the isoelectric point, or the pI, is just a pH value. It's the exact pH where a molecule is going to have an overall neutral charge. And so now that we know that, we can talk about how to calculate the isoelectric point of amino acids with non-ionizable R groups. And so the isoelectric point can actually be pretty easily calculated just by using the pKa values of a molecule. And the amino acid isoelectric point is really just a simple average. It's really just a simple average of the appropriate 2 pKa values. And that's what we said in our previous video too, that even if you're given multiple pKa values, the pI is always just going to be an average of 2 pKas. And so the good thing is that with non-ionizable R groups, these amino acids only have 2 pKas. So it makes it pretty easy to figure out which 2 pKas you need to average.

And so down here, what we have is the formula for the isoelectric point. And what you can see is that the isoelectric point is really just the average of the correct pKa values, the proper 2. So we have pKa₁ and pKa₂, and it's just going to be an average of these 2. And so in our example, it asks us to calculate the isoelectric point of valine. And recall that valine is one of the 13 amino acids that does not have an ionizable R group. Valine has a non-ionizable R group, which means it only has 2 pKa values, and the 2 pKa values are given to us. So all we need to do to calculate the isoelectric point is to take the average of the 2. So the isoelectric point is just going to be 9.62 + 2.322.

And so this comes out to 11.942. And if we type this into our handy dandy calculators, the answer comes out to be 5.97. And so what we can see is that the pI of valine is going to be 5.97, which matches up with option c, and all the other options are incorrect. So we can give c a check mark here to indicate that c is correct. And so we'll be able to get more practice calculating the isoelectric point of non-ionizable R groups, of amino acids with non-ionizable R groups in our next practice video. So I'll see you guys in those videos.

Draw the structure of Leu and determine its pI. pK _a1 = 9.6. pK_a2 = 2.4.

a. 7.59                                          

b. 6.0

c. 3.91

d. 5.5

• A. 7.59
• B. 6.0
• C. 3.91
• D. 5.5

At any pH below the pI, the population of amino acids in solution with non-ionizable R-groups will have:

• A. a net negative charge.
• B. a net positive charge.
• C. no charged groups.
• D. no net charge.
• E. positive and negative charges in equal concentrations.

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 specific pH at which the molecule has no net charge. At this pH, the positive and negative charges within the amino acid balance each other out, resulting in an overall neutral charge. This property is crucial for techniques like electrophoresis, which separates proteins based on their charge. The pI can be calculated by averaging the two relevant pKa values of the amino acid, particularly for those with non-ionizable R groups.

How do you calculate the isoelectric point of an amino acid with non-ionizable R groups?

To calculate the isoelectric point (pI) of an amino acid with non-ionizable R groups, you need to average the two pKa values of the amino acid. The formula is:

$\mathrm{pI}=\frac{{\mathrm{pK}}_{\mathrm{a1}}+{\mathrm{pK}}_{\mathrm{a2}}}{2}$

For example, if the pKa values of valine are 9.62 and 2.32, the pI is calculated as follows:

$\mathrm{pI}=\frac{9.62+2.32}{2}=5.97$

Why is the isoelectric point important in electrophoresis?

The isoelectric point (pI) is important in electrophoresis because it allows scientists to separate proteins based on their charge. At the pI, a molecule has no net charge and does not migrate in an electric field. By adjusting the pH to match the pI of a specific protein, scientists can isolate and study that protein without interference from other charged molecules. This makes pI a valuable property for biochemical analysis and purification techniques.

What is the relationship between the acidity/basicity of amino acids and their isoelectric points?

The relationship between the acidity/basicity of amino acids and their isoelectric points (pI) is that more acidic amino acids have lower pI values, while more basic amino acids have higher pI values. This trend occurs because acidic amino acids have more acidic pKa values, leading to a lower average pI, whereas basic amino acids have more basic pKa values, resulting in a higher average pI. Understanding this relationship helps in predicting the behavior of amino acids in different pH environments.

How does the isoelectric point affect the migration of molecules in an electric field?

At the isoelectric point (pI), a molecule has no net charge, meaning the positive and negative charges within the molecule balance each other out. As a result, the molecule does not migrate in an electric field. This property is utilized in techniques like electrophoresis, where adjusting the pH to the pI of a molecule allows for the separation and isolation of specific proteins or amino acids based on their charge. This non-migration at pI is crucial for accurate biochemical analysis.