Edman Degradation Reaction Efficiency - Video Tutorials & Practice Problems
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Edman Degradation Reaction Efficiency
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In this video, we're gonna talk about Edmond degradation reaction efficiency. So in our previous videos, we've said that Edmond degradation can only be used on small peptides, And here we're just reinforcing that same idea with a little bit more detail by saying that Edmond degradation is only practical for small peptides with less than about 50 amino acid residues. Now that might seem like a really limiting factor about Edmond degradation since most proteins in nature have way more than just 50 amino acid residues. But that's exactly why most proteins need to be cleaved down into smaller peptide fragments with less than 50 amino acid residues each in order for those proteins to be sequenced via Edmond degradation. But the question here is actually why? Why is it that Edmond degradation is only practical for small peptides with less than 50 amino acid residues? Well, it turns out the answer has to do with the reaction efficiency per cycle for Edmond degradation. And the reaction efficiency per cycle for most modern admin degradation sequinators is about 99%, which means that in each Edmond degradation cycle, 99% of the reactions or 99% of the peptides are going to react successfully really And so let's face it, a 99% reaction efficiency is a really high success rate and if you guys were to get a a 99% on your next test, I wouldn't be complaining. You wouldn't be complaining. We'd all be pretty happy and satisfied. But with admin degradation, 99% reaction efficiency means that in each Edmond degradation cycle, there's still gonna be about 1% of the reactions or 1% of the peptides that are going to fail to release their n terminal amino acid residue in the correct cycle. And so although 99% reaction efficiency seems really, really high, we have to remember that this is the reaction efficiency per cycle. And one Edmond degradation cycle reveals only 1 amino acid residue. And so if we have 50 amino acid residues in the protein, then we need 50 Edmond degradation cycles. And again, with each Edmond degradation cycle, 1% of the peptides fail to release their amino acid and so we get an accumulation of 1% of failed products with each cycle and so this accumulation of failed products with each cycle is really the reason why Edmond degradation is limited to small peptides with less than 50 amino acid residues. So just to clear up that idea, let's take a look at our example down below. And notice in this example, we have a pool of identical peptides on the left hand side and these are decapeptides because they have 10 amino acid residues. And they have an amino N on the far left and a carboxyl N on the far right. And notice that the N terminal amino acid residue is highlighted here in gold. And that's because after one round or one cycle of Edin degradation, this n terminal amino acid residue highlighted in gold is the one that's going to pop off of the chain and be identified as a PTH amino acid residue. And so, notice that the first cycle of Edmond degradation can be initiated with Phenyl isothiocyanate or PITC to initiate the first reaction. And then we can treat it with trifluoroacetic acid or CF 3 COOH to initiate the second reaction. And then, of course, we treat the released amino acid derivative with aqueous acid or H3O plus to initiate the 3rd reaction that generates that PTH amino acid final product that we identify. And so we know, this from our previous lessons and the result is that most of the peptides are gonna release their n terminal n terminal amino acid residue, and we can see that down below. Most of the peptides here have indeed released their n terminal amino acid residue and that's, indicated by these check marks here. But notice that not all of the peptides release their N terminal amino acid residue. So this peptide here failed to release its n terminal amino acid residue. Now in this diagram, it might seem that 1 out of 4 or 25% of the peptides are going to fail to release their amino acid residue. But in reality, it's only 1% of the peptides that fail. And so, it's not as bad as 25%, but even with 1% that's still capable of limiting admin degradation to small peptides. And the real reason is because notice that this peptide here is saying, whoops. Guess I'll just release it in the next cycle. And that's really the issue here. The fact that the next cycle is supposed to identify the second amino acid residue, not the first amino acid residue. And so, if this peptide releases its N terminal residue in the next cycle, it's really just contaminating the second cycle with unwanted PTH amino acids. And so essentially, these unwanted PTH amino acid side products will accumulate with each admin degradation cycle. And so if you have enough admin degradation cycles, you'll get a lot of side products that accumulate and ultimately, these side products are going to obscure the results and make it really, really difficult are going to require more Edmond degradation cycles. And with more Edmond degradation cycles, there are going to be more side products that accumulate. And again, more side products accumulating means that the results are going to be obscured and more difficult to interpret. And so again, we know that most proteins in nature are, exist, as being naturally long, so they have lots and lots of amino acid residues and they range from having several 100 up to several thousands of amino acid residues. But that means if we were to try to sequence those large proteins with Edmond degradation, we would need several 100 to several thousands of Edmond degradation cycles. And, again, that's a lot of Edmond degradation cycles And that's exactly why the solution to sequencing long proteins in nature is to cleave down those large proteins into smaller fragments before Edmond degradation. And that's exactly why we talked about all of those protein cleavage techniques in our previous lessons, such as, amino acid hydrolysis, chemical cleavage, and peptidases. And so, this here, concludes our lesson on Edmond degradation reaction efficiency. And in our next lesson video, we're gonna talk about cumulative yield and how the reaction efficiency can be used to calculate the cumulative yield. So I'll see you guys in that video.
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Edman Degradation Reaction Efficiency
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5m
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Video transcript
In this video, we're going to talk about how to use the Edmond degradation reaction efficiency to calculate the cumulative yield, which is something that your professor is likely gonna want you guys to do. And so we already know that cumulative yield can be calculated from the reaction efficiency. And really, all the cumulative yield is, is the relative amount of a very specific product that's obtained in a chemical reaction. And so with Edmond degradation, the very specific final product that's obtained and analyzed is the PTH amino acid. And so notice with this equation shown below, that it expresses the relationship between the Edmond degradation reaction efficiency, the number of Edmond degradation cycles, and the cumulative yield. And so the reaction efficiency per cycle raised to the power of the number of Edmond degradation cycles is equal to the cumulative yield. And so just for some context, accurate protein sequencing typically requires a high cumulative yield, usually greater than about 60%. And so a cumulative yield of 60% just suggests that 60% of the products of that Edmonds cycle are actually the correct PTH amino acid. And the remainder of the percentage, essentially 40%, are going to be unwanted PTH amino acid side products. And so recall from our previous lesson video that it's these unwanted PTH amino acid side products that can obscure the results. And so essentially, the goal is to keep this percentage of unwanted PTH amino acid side products as low as possible and to keep the cumulative yield percentage as high as possible. And so a biochemist can expect accurate protein sequencing with any combination of reaction efficiency and number of Edmond degradation cycles, that gives a cumulative yield greater than 60%. But any combination of reaction efficiency and number of Edmond cycles that yields a cumulative yield lower than 60%, a biochemist should be really cautious because inaccurate protein sequencing is at risk. And so that's something important to keep in mind. And so with our degradation procedure has a reaction efficiency of 99%, where 1% of each reaction cycle produces unwanted PTH amino acid side products. Calculate the total cumulative yield of the correct PTH amino acid immediately after the 50th Edmond degradation cycle. And so because it's asking us to calculate the cumulative yield, all we need to do is use our equation up above. And so notice that we're given the reaction efficiency of 99%, and 99% as a decimal is 0.99. And so if we take the reaction efficiency and raise it to the power of the number of Edmond degradation cycles, which is 50 in our example problem, then we can get our cumulative yield. And the cumulative yield, we can abbreviate with cy here for cumulative yield. And so if you take your calculator and you do 0.9 9 raised to the power of 50, you'll get an answer of 0.605. And this is our cumulative yield in a decimal format. Format. So if we want to convert it to a percentage, all we got to do is multiply it by a 100%, and that is going to equal 60 point 5%. And so this is the answer to our example problem. 60.5% is the cumulative yield here. And so notice that 60.5% is greater than 60%, which means that at with a reaction efficiency of 99%, at the 50th Edmond degradation cycle, we can expect to get accurate protein sequencing. And so, what you'll notice is that this is just barely making the threshold of 60%. It's literally just 0.5% higher. And so if we were to add just one more admin degradation cycle, by essentially changing our value of the admin cycles here to a value of 51, what you would see is that this here would generate a cumulative yield of about 59.9%. And this is a cumulative yield below our 60% threshold. And so what that means is that with, 51 Edmond cycles, that already we're starting to risk accurate protein sequencing. And it's possible that we may not get accurate protein sequencing with a 51st residue here. And so that's why typically biochemists like to use a threshold of 60% to make sure that the correct PTH amino acid is in high abundance. And so this here concludes our lesson on how to use reaction efficiency to calculate the cumulative yield. And in our next couple of practice videos, we'll be able to get more practice. So I'll see you guys there.
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Problem
Problem
Assuming 98% reaction efficiency, calculate the total cumulative yield of the correct PTH-amino acid at the 50th Edman degradation cycle.
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Problem
Problem
A) A peptide with the primary structure Lys-Arg-Pro-Leu-Ile-Asp-Gly-Ala is sequenced by the Edman degradation procedure. If each Edman cycle is 93% efficient, what percentage of the PTH-amino acids in the fourth Edman cycle will be PTH-Leu?
B) What percentage of the PTH-amino acids in the eighth Edman cycle will be PTH-Ala?