Specific Activity - Video Tutorials & Practice Problems
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concept
Specific Activity
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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 gonna have and the more function that we're gonna 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 2 different samples have the same exact amount of activity, that means that they're going to have equal amount of the target protein. And so down below in our example, we're gonna 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 1, 2, 3, 4, 5 of the red proteins, and flask number 2 has 1, 2, 3, 4, 5 of the red proteins as well. So, even though FLAS number 1 has more proteins in general, FLAS number 2 and FLAS number 1 have equal amounts of 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 2 samples have equal amounts of target protein, then that means that they're gonna have the same activity. And so what that means is that flas number 1 and flas number 2, because they have the same they have equal amounts of target protein, the red target protein, that means that they're also gonna have equal amounts of activity. So here, option c is gonna 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 flas number 2, even though it's a lot more pure than, the red protein in FLAS 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 gonna talk about specific activity in our next video. So I'll see you guys there.
2
concept
Specific Activity
Video duration:
5m
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Video transcript
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 specific activity. And when it comes down to it, all the specific activity is 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 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. And so it's important to note that specific activity actually increases during purification. And the answer to our question here, the way that biochemist actually can tell if they've successfully purified a its maximum level, and, when that specific activity its maximum level, and, when that specific activity does not increase anymore because it's constant, biochemists know that they have a pure protein. And so let's take a look at our example down below to clear up this idea of specific activity. And we're going to compare the specific activity of the red protein of interest in both of the flasks shown below. And so 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? Now, we already know from our previous lesson video that both Flask 1 and Flask 2 have equal amount of activity, and that's because they both have a total of 5 of the red target proteins of interest. And so because the activity is the same between both flasks, the only thing that's gonna be different is the total amount of protein. And it's clear to see that flask number 1 has a lot more total protein than flask number 2. And so a bigger number in this denominator is actually going to make the specific activity smaller. It's going to make it a fraction. And a smaller number in the denominator is gonna make the specific activity much, much larger. And because we're looking for the flask that a means a more purified protein. So because flas 2 has a greater specific activity, the red protein in FLAS number 2 is more pure than the red protein is in FLAS 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. And in the first column of our chart here, we have the purification technique or step. And 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. And 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. And so, recall that the target protein activity is the amount of the target protein. And 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. And notice that the specific activity is actually increasing with each purification step. And again, a greater specific activity means a more purified protein. And 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. Now, notice that the target protein activity is really the top number here of our activity in our equation, and the total amount of protein here is really the bottom number here of our equation. And, of course, the specific activity is going to be 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. And so really 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. And so, 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.
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Problem
Problem
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?