in this video, we're going to talk about how to determine the net charge of a peptide or protein. So the net charge of a protein is actually dictated by the net charges of all of its ionized herbal groups. And so recall that the way that we determine the ionization oven Ionized Herbal Group is just by comparing the p k a of the ionized well group to the ph of the solution. And so we're gonna be comparing the P. K s to the P H. Just like we did in some of our previous lessons and then also recall that on Lee, the very first and the very last amino acid residues in the chain have free Orion, Izabal, Alfa Amino or Alfa car boxes groups, respectively. And the internal amino acid residues lack Alfa amino or Alfa car boxful, ionized able groups. And so the last thing I want to leave you guys off with before we get to our example is the fact this idea that the net charge of a known poly peptide can Onley be estimated. You can Onley estimated unless you determine it experimentally. And the reason is because there is a unique micro environment for every single peptide. And so all the micro environment is is it's the immediate vicinity surrounding an atom or molecule. So it's the immediate area surrounding an atom or molecule, and the micro environment can actually shift. It can affect the polarity of the atoms, and it can actually shift the PK a values of amino acid residues by several units. And so when we go to compare the P K s to the P H, if the P K s are shifted by several units, that's going to affect the net charge. And so if that confused you a little bit, all I really want you guys to know is that I want you to double check and make sure that you're using the correct set of PK A values. So you wanna make sure that you're using the P K A values for amino acid residues and not for free amino acids. And so if you're professor uses the same set of PKK's for free amino acids and amino acid residues, that's totally fine. You're good to go, and you don't have to worry about anything. But if you're a professor expects you to use an entirely different set of P. K s for amino acid residues than for free amino acids. Then you need thio zone in a little bit more and make sure that you're using the right set of PKS. And that's all. I want you guys to double check. Alright, so that being said, let's go to our example. And in this example, it says toe estimate the net charge of the peptide at physiological pH, which is 7.4 and the peptide years Argentine history, a Spartak acid and glutamine. And then over here in this blue chart over here, what we have is the PK a values. And this is specifically for, um, you know, acid residues. And so I remember that we said that normally the car boxful group has a PK of about two in the ballpark of about two. But for amino acid residues noticed that the PK is 3.5, which is shifted 1.5 units and again, that can affect the charges when we go to try to determine them. And so again, if you're using the wrong set of PK is, then that could affect your calculation on the estimation of the charges, so just make sure that you're using the right set of PKS and we're gonna be using this table here for all of our, um, P K s for amino acid residues. Alright, So that being said, we're gonna estimate the net charge of this peptide, and we don't need to redraw the peptide. We just need to consider all of its ionized herbal groups. So let's go ahead and rewrite the peptide over here just so that we have a little bit more space and so we need to just consider it's ionized able groups. So we know Argentine over here is gonna have a, uh, amino group that we need to consider that's ionized able and Argentine, we know is one of the positively charged basic amino acids. So we know that it's our group we also need to consider to see if it's gonna be ionized or not. So is history into our group that we need to consider So I'm leaving these blank spaces here so that you know that these air, the ionized able groups that we need to consider ah Spartak Acid remembers a negatively charged, acidic amino acid. So we need to consider. It's our group, Aziz. Well, and then Glutamine does not have a nine Izabal our group. But it's on the end and we know that it's gonna have a nine invisible car boxful group. So Well, Leo, a spot here. So basically, we have 12345 ionized able groups that we need to consider for this peptide. And so we're gonna be comparing the Pete, the PKK's of each of these ionized able groups to the pH that's given to us of 74 So let's first start with the amino group on the far left over here. And so what we need to do is check our amino group on here and notice that our end terminus are amino group. Hold on, let me get rid of this color here. Notice that our amino group eyes right here the n terminus, and it has a PKF eight. And so ah, PK of eight is greater than the ph of 7.4. And when the PK is greater than the pH, that means the conjugate acid form predominates and so weaken draw the amino group in the kanji acid form. So it's gonna be NH three. Plus, it's just gonna have a positive charge. Now, for now, let's check. Argentines are group and Argentines are group is right here. So it has a PK of 12.5 and the PK of 12.5 is greater than the ph of 7.4. So that means again the conjugate acid is gonna predominate and we don't need to redraw. All of Argentines are group. I'm just gonna put a positive charge in here, so we know that we need to consider this positive charge. Now, let's do the same for history in and when we check history noticed that his sitting right here has a PK of six and the PK of six is actually smaller than the ph of 7.4. So what that means is that the conjugate base form is gonna predominate, and the conjugate base of history is actually a neutral charge. So it's gonna have a zero or a neutral charge here and then ask pretty or a Spartak acid. Uh, if we checked that over here, a Spartak acid has a PK of 3.9 notice it has a PKF 3.9 And so PKF 3.9 is smaller than the peak, the pH of 7.4. So what that means is that the conjugate base is going to predominate. The conjugate base form of a Spartak acid is charged, and it's gonna be negatively charged, so it's gonna have a negative charge. So you could see how you really need to know that our groups to be able to do this. And then what we could do is check the car box, a group in the car boxing group, the C terminus, which is, uh, right here appear. So the C terminus is 35 The PK of 3.5 is smaller than the ph of 7.4, which means that the conjugate base is gonna predominate. And that means that it's gonna have a negative charge. So it's gonna be C O minus negative charge. So now all we need to do is total the charges. So we've got a two positive charges over here, so that's a plus two. We've got a zero charge, so it's still plus two. Then we have a negative charge. So now we have toe deduct one. So now we're down to a plus one charge, and then we have another negative charge so that deduct another one. Now we're down to a zero charge. So now what this means is that the estimation of the net charge for this peptide at ph. 7.4 is going to be zero. It's gonna be B zero. And so you can see, um, that there's multiple steps here. You don't have to draw all the are groups. You just need to determine the net charge of each of its ionized able groups. And so, uh, a a c and D here are all incorrect. B is the correct answer. And so we'll be able to get some practice determining the Net charges of peptides and our practice videos, so I'll see you guys in those videos.