Specific Activity - Online Tutor, Practice Problems & Exam Prep

Activity measures the function and amount of a target protein, while specific activity indicates protein purity. Specific activity is calculated as the ratio of target protein activity to total protein amount. Higher specific activity signifies greater purity, as seen in protein purification techniques like affinity chromatography. As purification progresses, total protein decreases, but specific activity increases until it reaches a constant maximum, indicating a pure protein. Understanding these concepts is crucial for biochemists in assessing protein purification efficiency.

Specific Activity

In this video, we're going to begin our discussion on specific activity. But before we talk about specific activity, we're first going to talk about activity in general. And so activity is just a quantitative measure of both the function and the amount of a specific target protein of interest. And so the greater the activity, the more of the target protein we're going to have and the more function that we're going to get from the target protein. And so when biochemists are interested in purifying a target protein, they normally take the time to develop an assay for the target protein. And so an assay is any test or experiment that will measure both the presence, as well as the activity of a target protein. And so this assay can be performed at any point during our protein purification process, on just a small portion of the sample, so we don't need to use a lot of our sample. And we do this assay to check for the activity of the target protein, just to make sure that our target protein is even still there after we performed a purification technique. And so what's important to note is that if two different samples have the same exact amount of activity, that means that they're going to have an equal amount of the target protein.

And so down below in our example, we're going to clarify this idea of activity. And so, in our example, we're going to compare the activity of the red protein in both of the flasks below. So notice we have flask number 1 on the left and flask number 2 on the right, And these marble-looking things are representing proteins. And so notice that flask number 1 has a lot more proteins than flask number 2, But specifically for the red protein, which is our target protein of interest in this case, notice that flask number 1 has one, two, three, four, five of the red proteins, and flask number 2 has one, two, three, four, five of the red proteins as well. So, even though Flask number 1 has more proteins in general, Flask number 2 and Flask number 1 have equal amounts of the target protein, of the red target protein. And so this question over here asks, which flask has greater activity? Is it flask number 1, flask number 2, or do both flasks have an equal amount of activity? And so recall from our lesson up above that activity is a measure of both the function and the amount of a target protein. And if two samples have equal amounts of target protein, then that means that they're going to have the same activity. And so what that means is that Flask number 1 and Flask number 2, because they have the same they have equal amounts of target protein, the red target protein, that means that they're also going to have equal amounts of activity. So here, option c is going to be the correct choice. We can mark it as being the correct choice and cross off all the other options. So what you can see here is that activity will tell us the amount of the target protein. But what activity will not tell us is how pure our target protein is. So notice that the red protein in flask number 1 is not very pure at all because there are so many other different types of proteins. And the red protein in Flask number 2, even though it's a lot more pure than, the red protein in Flask number 1, the activity of both of these proteins is still exactly equal. And so how do biochemists determine if their target protein is pure? And that's exactly where specific activity comes into play, and we're going to talk about specific activity in our next video. So I'll see you guys there.

Specific Activity

So in our last lesson video, we said that activity is a measure of the amount of a target protein, but the activity alone does not tell a biochemist if their protein is pure. And so here we have a question and it asks, how can biochemists actually tell if they've successfully purified a protein? Well, it turns out the answer has to do with the specific activity, and that's because protein purity can be measured by a target protein's specific activity. And when it comes down to it, all the specific activity is just a ratio. It's the ratio of the target protein activity over the total amount of all of the protein that's present. And so looking at our example below, notice we have an equation for the specific activity. And really, all the specific activity is just the ratio of the target protein activity or the amount of the target protein, over the total amount of all of the protein that's present. It's important to note that specific activity actually increases during purification. And the answer to our question here, the way that biochemists can tell if they've successfully purified a protein is by observing its maximum level. When that specific activity reaches its maximum level, and when that specific activity does not increase anymore because it's constant, biochemists know that they have a pure protein.

Let's take a look at our example down below to clear up this idea of specific activity. We're going to compare the specific activity of the red protein of interest in both of the flasks shown below.

Notice the question below is asking, which flask has a greater specific activity? Is it flask number 1, flask number 2, or do both flasks have equal specific activity? We already know from our previous lesson video that both Flask 1 and Flask 2 have an equal amount of activity because they both have a total of 5 of the red target proteins of interest. Because the activity is the same between both flasks, the only thing that's going to be different is the total amount of protein. It's clear to see that flask number 1 has a lot more total protein than flask number 2. A bigger number in this denominator is actually going to make the specific activity smaller. It's going to make it a fraction. A smaller number in the denominator is going to make the specific activity much, much larger. And because we are looking for the flask that means a more purified protein. So because flask 2 has a greater specific activity, the red protein in flask number 2 is more pure than the red protein in flask number 1. And that's pretty clear to see here visually.

Now, over here on the right, we're going to change gears a little bit and talk about how specific activity relates to these protein purification techniques that we covered in our previous lesson videos. In the first column of our chart here, we have the purification technique or step. First, we have the crude cellular extract that results from protein extraction. Then we have the sample after salting out, ion exchange, size exclusion, and affinity chromatography, and then the sample after a second round of affinity chromatography. What you'll notice is that with each protein purification step, the volume of the sample is decreasing, the total amount of protein is decreasing, and the target protein activity is also decreasing.

Recall that the target protein activity is the amount of the target protein. That might seem like something that we don't want to happen. If we're trying to purify a target protein, why would we want the amount of the target protein to decrease? Well, it turns out that's something that we don't want to decrease. But it's inevitable with protein purification, and we're going to lose some of our target protein activity. Notice that the specific activity is actually increasing with each purification step. And again, a greater specific activity means a more purified protein. Notice towards the bottom that the specific activity is maximal, and it's also constant since it doesn't increase even after a second round of affinity chromatography. And so that means that we have a pure target protein at that point.

The target protein activity is the top number here of our activity in our equation, and the total amount of protein here is the bottom number here of our equation. The specific activity is then the specific activity over here. So essentially, if we take this yellow column right here and we divide that number by the total protein column here, we'll get the number, the specific activity number in this column here. The major takeaway that I want you guys to get from this video is that the larger the specific activity is, the more purified a protein is.

This concludes our lesson on specific activity, and we'll be able to get some practice utilizing these concepts in our next practice video. So I'll see you guys there.

A biochemist discovers and purifies a new enzyme and generates the purification table below.

A) Fill-in the table below with the specific activity of the enzyme after each purification procedure.

B) According to the data, which purification step was most effective to give the greatest relative increase in purity? ____________________________

C) Which purification step was least effective to give the lowest relative increase in purity? ____________________________

D) Based on the results in the table, should the biochemist be convinced that the enzyme is pure after step 6? Why?

Alright. So here's a 4 part practice problem that requires a bunch of calculations. So if you don't have your calculator, make sure you go grab it. It says a biochemist discovers and purifies a new enzyme and then generates the purification table shown below. Part a wants us to fill in the table with the specific activity of the enzyme after each purification procedure. Notice this column here has the specific activity and it's blank throughout the entire column. Specific activity is a measure of the purity of a protein and really, it's just a ratio. The specific activity recall is the ratio of the target protein activity to the total amount of protein. In order to get the specific activity, all we need to do is take the number in the target protein activity column and divide it by the number in the total protein column. So 4,000,000 divided by 20,000 gives us a specific activity of 200 for the crude cellular extract. For the 3,000,000, the next one, 3,000,000 divided by 12,000, gives us a specific activity of 250 for differential centrifugation. 1,500,000 divided by 4,000 gives us a specific activity of 375. If we do the same for the others, we'll end up getting 4,015,000 again. That is it for the specific activity and for part a. Now that we're done with part a, we can move on to part b. Part b down below says, according to the data, which purification step was most effective to give the greatest relative increase in purity. Notice down below, it's providing an equation for the relative purity of the technique, which is equal to the specific activity of the technique divided by the specific activity before the technique took place. If we go back up to our chart, notice we have a column for the relative purity of the technique. Because our first step here is the crude cellular extract that results from protein extraction, it's not really a purification technique, so it doesn't have a relative increase in purity. So we have, NA here. But differential centrifugation is our first protein purification technique. All we need to do is use this equation where we take the specific activity of the technique itself, divided by the specific activity that was present before the technique took place. So if we take 250 and divide it by 200, we'll get the relative purity for differential centrifugation. That comes out to 1.25. This number means that differential centrifugation increased the relative purity by a factor of 1.25. If we do the same for all of the other techniques, we'll take 375 divided by 250 to get 1.5. If we take 4,000 divided by 375, we'll get 10.66. If we take 15,000 divided by 4,000, we'll get 3.75. If we take 15,000 and divide it by 15,000, we'll get 1. Back in problem, part b, it's asking which one was most effective to give the greatest relative increase in purity. The greatest relative purity turns out to be 10.66, which corresponds with ion exchange chromatography in this particular example here. Down below in part b, we can put step number 4, ion exchange chromatography has a purity of 10.66. So, that is it for part b, and we can move on to part c. Part c is asking which purification step was least effective to give the lowest relative increase in purity. Going up to our chart, notice that the lowest relative purity is 1, and that surprisingly corresponds with affinity chromatography. Technically, our answer below for option c is going to be step 6, which is affinity chromatography. The reason why this is surprising is because in our previous lessons, we said that affinity chromatography was one of the more effective types of chromatography, not one of the least effective types of chromatography. If we take a look at our example chart here, the reason for why it appears to be the least effective is because notice that by the time affinity chromatography actually came into play, our protein was already pure.The reason we know that it was already pure is because notice that the specific activity reaches a maximal point and then remains constant by the time affinity chromatography came into play. That is why, affinity chromatography appeared to essentially not increase. When it asks, based on the results in the table, should the biochemist be convinced that the enzyme is pure after step 6? We can say, yes, they should be convinced that the enzyme is pure after step 6. The reason for why again has to do with the fact that the specific activity in our chart gets to a maximal point and then remains constant even after step number 6 is applied with affinity chromatography. Down below our answer is yes, and the reason for why is because the specific activity, which I'll abbreviate with SA here, is both maximal and constant. Essentially, that concludes our 4 part practice problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is the difference between activity and specific activity in biochemistry?

Activity measures the function and amount of a target protein in a sample. It indicates how much of the target protein is present and how well it performs its function. Specific activity, on the other hand, is a measure of protein purity. It is calculated as the ratio of the target protein's activity to the total amount of all proteins in the sample. Higher specific activity means a purer protein. As purification progresses, total protein decreases, but specific activity increases until it reaches a constant maximum, indicating a pure protein.

How is specific activity calculated in protein purification?

Specific activity is calculated using the formula:

$\frac{\mathrm{Activity} \mathrm{of} \mathrm{target} \mathrm{protein}}{\mathrm{Total} \mathrm{amount} \mathrm{of} \mathrm{all} \mathrm{proteins}}$

This ratio helps determine the purity of the target protein. As purification steps progress, the total protein amount decreases, but the specific activity increases, indicating a more purified protein. When specific activity reaches a constant maximum, it signifies that the protein is pure.

Why does specific activity increase during protein purification?

Specific activity increases during protein purification because the total amount of all proteins in the sample decreases while the activity of the target protein remains relatively constant. This results in a higher ratio of target protein activity to total protein amount. Each purification step removes more non-target proteins, thereby increasing the specific activity. When specific activity reaches a constant maximum, it indicates that the target protein is pure and further purification steps are unnecessary.

How do biochemists determine if a protein is pure?

Biochemists determine if a protein is pure by measuring its specific activity. Specific activity is the ratio of the target protein's activity to the total amount of all proteins in the sample. As purification progresses, specific activity increases. When it reaches a constant maximum and does not increase further, it indicates that the protein is pure. This is because all non-target proteins have been removed, leaving only the target protein in the sample.

What role does specific activity play in protein purification techniques?

Specific activity is crucial in protein purification techniques as it helps assess the purity of the target protein. During purification, techniques like affinity chromatography, ion exchange, and size exclusion chromatography are used to separate the target protein from other proteins. As these steps progress, the total protein amount decreases, but the specific activity increases. A higher specific activity indicates a more purified protein. When specific activity reaches a constant maximum, it signifies that the target protein is pure, and no further purification is needed.