Leah here from leah4sci.com and in this video

we’re going to look at finding charge and isoelectric point for a peptide, where the

peptide can be anywhere from a dipeptide, two bound amino acids, tripeptide, three or

poly- many. In the last video we looked at how to find the charge of a single amino acid

by determining at that given pH if the carboxy group was protonated or not. The amine group

and the R-group if it has an ionizable side chain. Once we found the charge, we were able

to add them all up and get a net charge at that specific pH. Where you’re looking at a peptide you have

to take all ionizable groups into account, both parent and side chain add up all the

charges and that will give you the net charge. For example, say we’re asked to find the charge

of Gly-Asp at a pH of 8. I’ll show you the long way and then the short way. The long

way is to draw out your dipeptide. In this case your first amino acid N to C would be

glycine and the side chain for glycine is simply a hydrogen atom. For the peptide bond

we have dehydration synthesis, so we remove the oxygen. We place a single bond between

the carbonyl carbon and the incoming nitrogen that now has only one hydrogen. And of course

the side chain for aspartic acid which is the CH2 bound to a carboxy group. Then we

have to figure out if each ionizable side chain is protonated or deprotonated. By comparing

the pKa to the pH. On the MCAT, you’re probably not getting a pKa table so I recommend memorizing

these numbers. For an alpha amine, memorize 10. For an alpha carboxy memorize 2, for aspartic

acid and glutamic acid memorize 4. Now we’ll compare the pH to the pKa for each

individual group. Remember, the one that’s higher wins a proton. If the pH is higher

than the pKa, the pH wins one for the solution and the molecule is deprotonated. If the pKa

is higher than the pH, the pKa wins one for the molecule and the molecule is protonated.

pKa 10 is greater than a pH of 8, we win one for the molecule making it NH3+. Looking at

the aspartic acid side chain, pKa 4 is less than the pH that means the pH wins one for

the solution. We have a deprotonated side chain and a charge of negative one and same

thing for the alpha carboxy where pKa 2 is less than pH 8. Once again, negative charge.

If we add up the charges, we get plus one for the alpha amine, minus one for the alpha

carboxy, minus one for the side chain, for a net charge of negative one. This was the

long way, and on your MCAT you do not wanna waste time drawing out skeletons, writing

out side chains, it’s too much time. Instead write out your three or one letter name to

know which amino acid you’re looking at then simply add your ionizable side chains. Glycine

has a nitrogen, it does not have an ionizable R-group. Aspartic acid has a carboxy and an

alpha carboxy, so that is all we’re looking at. And then we do the same thing. 10 is higher

than the pH, plus one. 4 is less than the pH minus one. 2 is less than the pH minus

one and we quickly add it up. Plus one minus one cancels, negative one is the net charge. Say we’re asked to find the charge of this

dipeptide at a pH of 3. We’ll do the same thing and look at each group one at a time.

The amine group with the pKa of 10 is greater than the pH and that means it wins one for

the molecule, NH3+. The side chain with a pKa of approximately 4 is greater than the

pH, but not by much. This is where we’re in a buffer zone but for the sake for a quick

net charge, we’re going to estimate where the pKa is higher than the pH wins a proton

for the molecule giving us a neutral protonated group. But the side chain with the pKa around

2 is less than the pH, that means it’s deprotonated for a charge of negative one. If we add it

up, we get a total charge of plus one for the nitrogen, plus zero for the side chain

carboxy, minus one for the alpha carboxy for a net charge of zero. Having a net charge

of zero tells us that we’re somewhere near the isoelectric point at a pH of 3. In the

last video, we saw that to find the isoelectric point with two pKa values we simply take the

average of the two. When you have more it gets a little tricky. When you have multiple

amino acids bound that�s when it gets even trickier. There are many way to approach it

but on the MCAT you need something quick, you need something easy and you definitely

don’t want any guess. So here is how I approach the isoelectric

point of a polypeptide when you potentially have multiple pKa values to take into consideration.

Instead of doing the guess and check method where you guess where the pH is approximately

zero and then find the pKa above and below the average, I like to start with knowing

exactly which two pKa values to choose and I find that by following this step. Number

one, protonate all ionizable groups. So we�ll forget that the pH is 3 and simply start again.

A protonated alpha amine has NH3+, protonated carboxy is zero in both cases. The second

thing we do is find the net charge, and this is going to be your maximum charge because

every time you deprotonate your charge will go down. In this case we have a plus one for

the amine, two zeros for the carboxy, for a net charge of plus one. Step three is tricky

so really try to understand. If your charge is plus one, ask yourself, how many units

away from zero am I? And line up the pKa values in order because the lower the pKa, the earlier

it will be deprotonated. If we line them up here, we have approximately 10, approximately

4, and approximately 2. Lining them up in order, we have pKa values of 2, 4, and 10.

If the pH is under the pKa, meaning it’s lower than the lowest pKa value, that’s when we

have the fully protonated molecule, so this structure would exist at a pH of something

less than 2. For example, pH of 1, pH of a 1/2. So at a pH of 1, the charge we already

determined to be +1. Everytime you raise the pH to go above the next pKa, you’re going

deprotonate one more group. Because if we raise the pH to say, 3, then the ionizable

group that has the pKa of 2 is going to lose its proton. But instead of calculating it

all over again, simply drop the charge by one. Between a very low pH and pKa 2, we have a

charge of plus one. If we raise the pH to in between the next two units, the charge

drops by one and that means we have a charge of zero. If we raise the pH in between the

next pKa, meaning we raise it above a pH of 4 which is in between 4 and 10, the charge

should drop by one more. It would be negative one. If we raise the pH to above the highest

pKa, we have a fully deprotonated molecule and this is where you’re going to have the

lowest charge of -2. Let’s prove that really quick before we go back to step 3. And see

what happens if we have a pH of, let’s use this one as 7, and we�ll make this one 12.

At a pH of 12, we’re going to deprotonate nitrogen, giving me NH2 with a charge of zero.

We will deprotonate the carboxy for a minus one and deprotonate the carboxy for a minus

one. When we add it up we have zero, minus one and minus one for a net charge of -2,

as predicted. I want you to try this for every pH to prove to yourself that the trick works

and then remember this. Step 3 is to find the pKa before and after the zero charge.

In this case we have a charge of zero at pH 3 but it could’ve been 2 something, it could’ve

been 3 something. In this case, we’re doing an estimate so it will be 3. What we’re looking

at is where do I get a charge of zero? In between which two pKa values. It’s in between

the alpha carboxy and the carboxyl group of aspartic acid, and so we take the average

of those two pKa values. In this case we have 2 plus 4, the average over two which gives

me 6/2, that’s 3 and that’s what we estimated. If you’re given pKa values, the numbers will

not be as clean because you’ll be rounding from values off a table, but the idea is still

the same. Let’s try this again by looking at the tripeptide

made by glycine, glutamic acid and histidine. We’re not gonna draw out the full polypeptide,

instead we’ll simply look at which ionizable groups we have. Remember, you have to know

your amino acid side chains so you have to know what we’re looking at here. Lysine is

the N-terminus and has an alpha amino. Lysine is also a basic amino acid with a nitrogen

that has a pKa of about 11 which you do need to memorize. Glutamic acid has a carboxylic

group with a pKa to memorize of approximately 4. Histidine has an aromatic ring with an

ionizable nitrogen at a pKa of about 6. It also has an alpha carboxy with a pKa about

2. So let’s go ahead and find the PI. Step one, protonate everything. NH3+, N we’ll just

put an H+ you don’t have to worry about the exact specifics how many hydrogens, it doesn’t

matter. A neutral carboxylic acid and a neutral carboxylic acid. Plus one for a protonated

nitrogen. Step 2 we’re going to find the net charge, that’s plus one for the alpha amine,

plus one for lysine, plus zero for glutamic acid, plus one for histidine and plus zero

for the alpha carboxy. For a net charge of +3. We have a fully protonated molecule and

this is going to be your maximum positive charge that you�re going to get on this

molecule. Step 3, we’re going to find the pKa before and after the zero charge. What

does this tell me? If I have a maximum charge of positive three, to get to zero I need to

go up to positive two, positive one, and zero. That means I need to jump a couple of pKa

units. That means I need to cross them out one at a time. If I’m at positive three to

start, to jump up to positive two, I have to get past the lowest pKa, that would be

2. To get to plus one, I have to go past the

next one which is 4, to get to zero I have to go past 6 to the next pKa, so that’ll be

between 6 and the next one which in this case is 10. If you didn’t follow along as I was

talking, write it out, get comfortable seeing it and then try to do it in your head. To

write it out we’re simply going to list all the pKa values in order. 2 for the alpha carboxy,

4 for the side chain carboxy, 6 for histidine, 10 for the alpha amine and 11 for lysine.

If the pH is below the first pKa value we have a charge of plus three going between

2 and 4 would give me plus two. Going between 4 and 6 would give me plus one. Going between

6 and 10 would give me zero, and that means 6 and 10 are the pKa values to average. And

so I set up my equation, and so the PI is equal to 6+10 over 2. 6 and 10 is 16, divided

by two is 8 and the PI for this molecule is equal to 8. Perhaps you’re already with me but I suspect

that you’re potentially confused and want to see this drawn out again. So let’s try

one more example by finding first the charge of the polypeptide �CHARGE� at different

pHs and then going back and applying the trick to verify that the PI trick works. First thing

we need to figure out is which amino acids are we referring to when we say CHARGE. C

gives me Cysteine, H gives me histidine, A is alanine, R is Arganine, G Glycine and E

glutamic acid. We’re not gonna draw them out, we’re not gonna fill in all these side chains,

all we’re doing is adding ionizable groups. We’ll start with an alpha amine on cysteine.

Cysteine has an ionizable side chain with an SH. Histidine has an ionizable side chain

with nitrogen. Alanine does not, I just put an x to remind me there’s nothing there, but

it still saves me time from having to draw it out. Argenine, �arg� it’s complicated

but all we’re looking at is nitrogen. Glysine is just a hydrogen so we’ll put an x because

it’s not an ionizable side chain. Glutamic acid has an R-group carboxy but it also has

the alpha carboxy. We’ll use estimated pKa values that you have to memorize. If you’re

not comfortable with them, go back to my amino acid tutorial which has a table of all the

values. You can find them on my website along with this entire amino acids tutorial series,

practice quiz and cheat sheet leah4sci.com/aminoacid. The pKa values to memorize is 10 for the alpha

amine, 8 for cysteine, 6 for histidine, 12 for argenine, 4 for glutamic acid side chain,

and 2 for the alpha carboxy. If we follow the earlier trick, the first step was to protonate

everything and get the charge. So let’s go ahead and protonate everything. Find a lower

pH and prove that the charge is what we predicted. If we protonate the nitrogen, we get NH3+,

sulfur is neutral, NH on histidine is positive, NH on argenine is positive. OH on the carboxylic

acids are neutral for each one. If we add them up, we get plus one for the alpha amine,

plus one for histidine, plus one for argenine, zeros for everything else and a net charge

of +3. This would happen at a pH lower than the lowest pKa which in this case is 2. So

let’s see what happens when this solution has a pH of 1. If the pH is 1 and the pKa

is greater than the pH, the pKa wins one for the molecule and it will be protonated. 10

is greater than 1, protonate nitrogen, 8 is greater than 1, protonate sulfur it’s neutral.

6 is greater than 1, protonate nitrogen, 12 is greater than 1, protonate nitrogen. 4 and

2 are both greater than 1, protonate the carboxy for a neutral group. We add it up and verified

that yes we have three positives, three zeros for a net charge of plus three. Let’s see

what happens if we raise the pH to 4. 10 is greater than 4, we protonate nitrogen. 8 is

greater than 4, we protonate sulfur. 6 is greater than 4, protonate nitrogen. 12 is

greater than 4, protonate nitrogen. 4 is equal to 4 and this is where we get half protonated,

half deprotonated so we’ll say half hydrogen, and that gives me a charge in between protonated

and deprotonated. Protonated would be neutral, deprotonated would be negative one so we get

negative �. Remember, when the pH is at a pKa, you’re in the buffer zone and that’s

half protonated, half deprotonated. And finally, 2 is less than 4 which means

it’s deprotonated for a charge of negative one. Adding the charges we have plus one,

plus two, plus three, minus one, and minus a half. This gives me a net charge of positive

1 � or 1.5. Let’s see what happens if we raise the pH to 7. 10 is greater than 7, protonate

nitrogen. 8 is greater than 7, protonate sulfur. 6 is less than 7, deprotonate nitrogen give

it electrons, keep it neutral. 12 is greater than 7, protonate nitrogen. 4 and 2 are less

than 7, deprotonate each one for a negative charge, this gives me a net charge of plus

one, plus one, minus one, minus one. At pH seven we have plus two minus two which is

zero that means the isoelectric point should be somewhere in that zone, but we’re not done.

Let’s try this one more time at a pH of 10. PH of 10 is equal to the pKa 10, that means

half of the alpha amine will be protonated and half will be deprotonated. The half that’s

protonated is going to give me a charge of plus one. The half that’s not protonated is

going to give me a charge of zero and that means, the charge will be plus �. PH 10

is greater than pKa 8. Sulfur is deprotonated for a charge of minus one. 10 is greater than

6 giving me a neutral nitrogen. 10 is less than 12 so we protonate the positive argenine,

10 is greater than 4 and greater than 2 so we deprotonate the carboxy groups giving each

one a negative charge. If we add up the charge, we get plus a half, minus one, plus one, minus

one, minus one. Notice I only focused on the charged groups because the neutral groups

are neutral. This gives me a net charge of negative 1.5 or negative 1 �. Just to complete

this exercise, let’s look at a pH of 13 knowing that it’s greater than every pKa on this molecule.

At a pH of 13, every side chain will be deprotonated because 13 is greater than all of the pKa

values here. That means nitrogen will be neutral, sulfur will be deprotonated, negative one,

nitrogen is neutral, nitrogen is neutral. Each carboxy is deprotonated and negative

one. The total charge will be a negative sulfur and two negative carboxy groups for a net

charge of minus three. Do you see what we did here, we proved the charge a different

pH values. Now I wanna show you what happens when we actually line up the pKa values in

order, because lining up the values will simply be a shortcut for what we did. We start with

the lowest pKa value, 2 for the alpha carboxy and then increase. 4 for glutamic acid, 6

for histidine, 8 for cysteine, 10 for the alpha amine and 12 for argenine. If we take

the pH below the lowest value, everything is protonated. And the groups that are protonated

to give me a positive charge are nitrogens. That means the alpha amine, nitrogen on histidine

and argenine for a net charge of plus three as we proved when we solved the pH of 1. Now the pattern, we expect the charge to go

down by one every time we jump above a pKa value. So if we jump between 2 and 4, we expect

it go down, that would be plus two, plus one, zero, negative one, negative two, negative

three. Let’s see if this pattern matches up with any of the numbers we predicted. At pH

4 we said one and a half and 4 is in between plus two and plus one, well that’s the middle

of two and one, one and a half. At pH 7 we predicted a charge of zero. 7 is between 6

and 8, that would be zero. At pH 10 we estimated negative 1.5, we predicted a charge of negative

1 under pH 10. A charge of negative 2 over pH 10 and what�s in between negative 1 and

negative 2? Negative 1.5. Finally when we go above the pH of the highest pKa we get

a charge of negative 3. We have already predicted that somewhere near pH 7 we expect a net charge

of zero, the isoelectric point but if you didn’t go through all of this you would still

get there because doing this trick will show you that somewhere in between 6 and 8 is where

you would get your isoelectric point and you simply setup your equation, PI is equal to

pKa one plus pKa two, the one that is directly above and below zero, so if zero is between

6 and 8, we have to use 6 and 8, divide that by two, 6 and 8 is 14 over 2 is 7 and that’s

what we’ve already predicted. If you were given specific pKa values your calculations

would be slightly different but very close to seven as we predicted here. For more practice

on this be sure to check out the amino acid quiz link below. Be sure to join me in the

next video where we look at different simple reactions involving amino acids as you�re

required to know for the MCAT. Remember you can find this entire video series along with

the amino acid practice quiz and cheat sheet on my website leah4sci.com/aminoacids

Omg this was the best explanation ever. It saved my ass on the exam. Thank you so much

Thank you Mam

Great job๐๐๐ฅ๐ฅ

just completed my assignment in few seconds because of You (This video) ๐

Okay so the way I learned this was that you take the average of the pKas where the amino acid (or peptide) has a net charge of +1/2 and -1/2 to determine the pI. Would this not make it more accurate than taking the avg of pKas at a net charge of +1 and -1?

This video is very helpful. More explanation than what I learned from my lecture. Thank you so much.

Thanks @LEAH4SCIMCAT

thank you!!! i finally get it!!

This is so helpful. Thank you so much!

You carried me through organic chemistry, and now you are carrying me through biochem. Thank you, you're the best!

Spring 2019, final quarter in the enduring Ochem series. You have enlightened my knowledge and your tutorials are a great supplement to my lecture notes. Thank you so much Leah it's been a great year following your channel. BIOCHEM here I come!

thanks verry much

We owe you money for this video thank you๐

I love you, thanks a lot!

Thank you Leah! You're the best. ๐

ABSOLUTELY AMAZING! THE BEST TEACHER EVER!!!

THIS WAS SO HELPFUL.THANK YOU SO MUCH!