Give the order of elution of the following peptides when using...

Question

Give the order of elution of the following peptides when using cation-exchange chromatography at pH 7.2.

Peptide #1: A-D-G-H-E. Peptide #2: K-L-M-R-A. Peptide #3: M-D-L-I-V. Peptide #4: I-L-R-P-M.

Order of Elution: _______________, _______________, _______________, _______________

(1 ^st to elute) (Last to elute)

Accepted Answer

Alright. So this practice problem wants us to give the order of evolution of the following peptides when using cation exchange chromatography at pH 7.2. And we're given the sequences for each of these 4 peptides and these blank spaces below where we can provide the order of elution of these 4 peptides from the first to elute from the column to the last to elute from the column. And so we know that cation exchange chromatography separates proteins based on the magnitude of their net charges. But notice that we're not actually given any of the net charges for any of these peptides, which means that we can break this practice problem up into 2 general steps. And in the first step, step number 1, we're going to estimate the net charges for each of the predominant structures for each of these peptides. And in step number 2, we're going to use the estimated net charges from step number 1 to predict the order of elution of these peptides from the cation exchange chromatography column. And so the good thing here is that for step number 1, we already know how to estimate the net charges of peptides from our previous lesson videos. And in the past when we did this, we separately compared each of the ionizable group p k a's to the pH of the solution. Now we have the pH of the solution as 7.2, but notice we're not given any of the p k a's for any of the ionizable groups, which means that you either had to have memorized all of the p k a's for all of the ionizable groups, which your professor is not likely going to expect you guys to do, or you have to use some kind of amino acid p k a chart or table. And you guys are more than welcome to use an amino acid p k a chart or table to estimate the net charges of each of these peptides like we did in our previous practice problems. But the reason that I didn't provide a p k a charter table for this practice problem is because we really don't need one, and that's because we're using cation exchange chromatography at pH 7.2, which is right around physiological pH, which means that the ionizable groups are going to have the ionizable states as we originally memorized them when we first introduced the amino acids. And so we know, for instance, at physiological pH, each of these peptides is going to have an ionized amino group on the n terminal end. And so we can put a positive charge to represent the amino group that is ionized at physiological pH for each of these peptides. And, in a similar fashion, we know that on the carboxyl end, there's gonna be an ionized carboxylate group at physiological pH for each of these peptides as well. So we can put a negative charge here to represent that. And so now that we have the amino and carboxyl groups taken care of for each of these peptides, we now only need to focus on the R groups. And recall the mnemonic that helps us memorize the amino acids with ionizable r groups is just yucky crazy dragons eat knights riding horses. And so if it's not one of these 7 amino acids, then its r group is not ionizable. It's not gonna contain a charge and it's not gonna contribute to the net charge of the peptide. So that means that we can essentially not worry about it if it's not part of this group. And so looking at our first amino acid residue, alanine, notice it's not part of this group, so we don't have to consider it in the net charge, and we can move on to the next one. And the next one here is D, which is for aspartic acid, and that is part of our mnemonic, and we know that at physiological pH of 7.2, aspartic acid's R group has a negative charge, so we can put a negative charge here. Now glycine is not part of our mnemonic, so we can skip it. Histidine is part of our mnemonic, and we know that at physiological pH, it's going to have a positive charge on it. And then glutamic acid here is going to be part of our mnemonic as well, and it's going to have a negative charge at physiological pH. And so now all we need to do to get the net charge of this peptide is to sum up all of these charges. So we've got 3 negative charges and 2 positive charges, which comes out to a total net charge of negative one. Now moving on to peptide number 2, k is part of our mnemonic down below, and so we know it's gonna have a positive charge at physiological pH. Now leucine and methionine are not part of our mnemonic, so we can skip those, but arginine is, so it has a positive charge, so we can go ahead and include that. And then alanine is not part of our mnemonic, so essentially here we have 3 positive charges and only one negative charge, so the net charge on peptide number 2 is going to be positive 2. Now with peptide number 3, we have methionine, which is not part of our mnemonic down below, and then we have aspartic acid, who is part of our mnemonic, and we already did it earlier, we know it has a negative charge. And then notice that leucine, isoleucine, and valine are not part of our mnemonic, so we can skip those r groups. So essentially, we have 2 negative charges and one positive charge, which means that we have a total net charge of negative one on peptide number 3. And then with peptide number 4, notice that isoleucine and leucine are not part of our mnemonic, so we can skip those. And arginine, we already did earlier, we know that it has a positive charge, so we can put a positive charge on arginine here. And then proline and methionine are not part of our mnemonic, so we can skip those. So essentially, we have 2 positive charges over here and one negative charge. So our total net charge for peptide number 4 is plus 1. Alright. So now we have finished step number 1. We've estimated the net charges. So we can go ahead and give step number 1 a check here. So now all we need to do is use the estimated net charges from step number 1 to predict the order of elution from the cation exchange chromatography column. And recall that cation exchange chromatography separates, or is used to collect and purify proteins that are positively charged. And so the positively charged proteins are gonna be the ones that get stuck in the column and move really, really, really slowly through the column. And so the positively charged proteins are gonna be the ones to elute last, and the ones that have a greater positive charge are gonna be the ones to elute last from the column. And so notice that peptide number 2 has the greatest positive charge of plus 2, so it's gonna be the one to elute last. So we can put it as eluting last, peptide number 2. Now, peptide number 4, notice, has the next greatest positive charge, so it's going to be the second to last here to elute. And then what you'll notice is that both peptide number 1 and peptide number 3 have identical charges, it seems, and, they are both negative one. And so the next step that we're going to need to realize is we're going to have to take into account this note here that we should always take into account, and that's the fact that histidine's p k r is pretty close to physiological pH. And because we're using pH of 7.2, which is close to physiological pH, we need to take that into consideration. So histidine's PKR is 6.0. And so you're not expected to memorize all of the PKRs, but this is definitely a good PKR to know for histidine. And so what you'll know is that histidine here, because it has a physiological, because it has a pKR of 6 that is pretty close to the pH of 7.2, it's actually going to have a partial charge at a near physiological pH. It's going to be partially charged. And so yes, it is going to have a positive charge, but it's going to have a partial charge. So let's just put a plus 0.5 here, just so that we know that it's going to be a partial charge. And so what that means is when we total the net charge, it's really going to be more like a negative 1.5 charge instead of a negative one. And so, now when we do that and take that into consideration here, we can pretty clearly see that peptide number 1 is actually going to be the first to elute because it has the greatest negative charge. And remember, in a cation exchange chromatography column, the negatively charged proteins elute first, and the greater the negative charge, so the greater than the magnitude of the negative charge, the earlier it will come out. So that means that peptide number 1 will be the one to elute first, and then of course, peptide number 3 will be the one to elute at this position here. And so what that means is that this here is the answer to our problem, and that concludes this, challenging practice problem, and I'll see you guys in our next video.

Struggling with Biochemistry?

Give the order of elution of the following peptides when using cation-exchange chromatography at pH 7.2.
Peptide #1: A-D-G-H-E. Peptide #2: K-L-M-R-A. Peptide #3: M-D-L-I-V. Peptide #4: I-L-R-P-M.

Order of Elution: _______, _, _, _______
(1 ^st to elute) (Last to elute)

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