In this video, we're going to talk about Edman degradation reaction efficiency. So in our previous videos, we've said that Edman 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 Edman 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 Edman 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 Edman degradation. But the question here is actually why? Why is it that Edman 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 Edman degradation. And the reaction efficiency per cycle for most modern Edman degradation sequinators is about 99%, which means that in each Edman degradation cycle, 99% of the reactions or 99% of the peptides are going to react successfully. And so let's face it, a 99% reaction efficiency is a really high success rate, and if you guys were to get 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 Edman degradation, 99% reaction efficiency means that in each Edman degradation cycle, there's still going to 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 Edman degradation cycle reveals only 1 amino acid residue. And so if we have 50 amino acid residues in the protein, then we need 50 Edman degradation cycles. And again, with each Edman 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 Edman 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 Edman 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 Edman degradation can be initiated with Phenyl isothiocyanate or PITC to initiate the first reaction. And then we can treat it with trifluoroacetic acid or CF3COOH to initiate the second reaction. And then, of course, we treat the released amino acid derivative with aqueous acid or H3O+ to initiate the third 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 going to release their 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 Edman degradation to small peptides. And the real reason is that 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 Edman degradation cycle. And so if you have enough Edman 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 Edman degradation cycles. And with more Edman 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 exist as being naturally long, so they have lots and lots of amino acid residues and they range from having several hundred up to several thousands of amino acid residues. But that means if we were to try to sequence those large proteins with Edman degradation, we would need several hundred to several thousand Edman degradation cycles. And, again, that's a lot of Edman 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 Edman 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 Edman degradation reaction efficiency. And in our next lesson video, we're going to 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.
Edman Degradation Reaction Efficiency - Video Tutorials & Practice Problems
Edman Degradation Reaction Efficiency
Video transcript
Edman Degradation Reaction Efficiency
Video transcript
In this video, we're going to talk about how to use the Edman degradation reaction efficiency to calculate the cumulative yield, which is something that your professor is likely going to want you guys to do. We already know that the cumulative yield can be calculated from the reaction efficiency. The cumulative yield is the relative amount of a very specific product that's obtained in a chemical reaction. With Edman degradation, the very specific final product that's obtained and analyzed is the PTH amino acid.
Notice with this equation shown below, it expresses the relationship between the Edman degradation reaction efficiency, the number of Edman degradation cycles, and the cumulative yield. The reaction efficiency per cycle raised to the power of the number of Edman degradation cycles is equal to the cumulative yield. For context, accurate protein sequencing typically requires a high cumulative yield, usually greater than about 60%. A cumulative yield of 60% suggests that 60% of the products of that Edman cycle are the correct PTH amino acid. The remainder of the percentage, essentially 40%, are going to be unwanted PTH amino acid side products.
Recall from our previous lesson video that these unwanted PTH amino acid side products can obscure the results. 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. A biochemist can expect accurate protein sequencing with any combination of reaction efficiency and number of Edman degradation cycles that gives a cumulative yield greater than 60%. However, any combination of reaction efficiency and number of Edman cycles that yields a cumulative yield lower than 60% should be approached with caution because inaccurate protein sequencing is at risk.
That's something important to keep in mind. 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 Edman degradation cycle. Since it's asking us to calculate the cumulative yield, all we need to do is use our equation from above. Given the reaction efficiency of 99%, and as a decimal, this is 0.99.
If we take the reaction efficiency and raise it to the power of the number of Edman degradation cycles, which is 50, then we can get our cumulative yield. The cumulative yield, abbreviated as cy, can be calculated using: cy 50 = 0.99 50 If you calculate this, you'll get an answer of 0.605, or 60.5% when converted to a percentage. This exceeds the 60% threshold, suggesting that with a reaction efficiency of 99% at the 50th Edman degradation cycle, we can expect accurate protein sequencing. Note that 60.5% is just barely above the threshold. Adding one more Edman degradation cycle, changing the cycle number to 51, the cumulative yield becomes approximately 59.9%, below our 60% threshold.
What this means is that with 51 Edman cycles, we're starting to risk accurate protein sequencing. It's possible that we may not get accurate protein sequencing with the 51st cycle. Typically, biochemists use a threshold of 60% to ensure the correct PTH amino acid is in high abundance. This concludes our lesson on how to use reaction efficiency to calculate the cumulative yield. In our next couple of practice videos, we'll be able to get more practice. I'll see you guys there.
Assuming 98% reaction efficiency, calculate the total cumulative yield of the correct PTH-amino acid at the 50th Edman degradation cycle.
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?