# isoelectric point question!

#### docntrainin

What is the isoelectric point for cysteine?

Information given in table:

pKa COOH (1.9) pKa NH3 (10.3) pKa R group (8.4).

Answer: 5.2. The isoelectric point is found by averaging the pKas that give a +1 charge to the molecule and a -1 charge to the molecule. Thus from the table values, pI = 1.9+8.4/2 = 10.3/2 = 5.15.

I understand that the isoelectric point is where zwitter ion exist and that it is where the +1 -1 charge is aka a neutral molecule, but how did they know which pKas to use? I just averaged the pKas of COOH and NH3. How did they know to use pKa of R group and pKa of COOH.

So confused! Thank you

#### tn4596

7+ Year Member
because the R group have lower pka than NH3, therefore it will be the first functional group to lose the hydrogen as pH increase, giving rise to the -1 overall charge on the amino acid. (the NH3 doesnt become NH2 until the pH exeed 10; SH group on Cys become S- when pH exeed 8).
Best way to do isoelectric point is to draw the aminoacid/peptide as pH increase like how they do it here
http://wwwchem.csustan.edu/chem4400/aapi.htm
then find out which functional groups change as the structure went from -1 to 0 to +1 overallcharge. you don't have to draw out the entire things to do this, all you need is the charges on each functional groups

Last edited:

#### docntrainin

because the R group have lower pka than NH3, therefore it will be the first functional group to lose the hydrogen as pH increase, giving rise to the -1 overall charge on the amino acid. (the NH3 doesnt become NH2 until the pH exeed 10; SH group on Cys become S- when pH exeed 8).
Best way to do isoelectric point is to draw the aminoacid/peptide as pH increase like how they do it here
http://wwwchem.csustan.edu/chem4400/aapi.htm
then find out which functional groups change as the structure went from -1 to 0 to +1 overallcharge. you don't have to draw out the entire things to do this, all you need is the charges on each functional groups
sorry, this is probably a stupid question but from that site, what does it mean by: To find the pI, average the two pKa values on either side of the neutral form of histidine.

#### tn4596

7+ Year Member
sorry, this is probably a stupid question but from that site, what does it mean by: To find the pI, average the two pKa values on either side of the neutral form of histidine.
You should know that the pkA of all COOH is 2 and NH3 is 10 for all amino acids. so you can deduct that the other pka is the pkR. (pkA of the side chain)
taht being said the picture on that website is lacking the labelling for pkA. The principle is simple, lets say when the overall charge is +2, as it is changing from being +2 to +1, the pH have to be higher than a certain value for the OH to become O-, that value is the pkA of COOH group, which is 2 (you can see why if you review acid-base equilibrium and definition of pka). you can use this to find out that from +1 to 0, it is the R side chain (NH+) becoming NH, that cause the change in overall charge. So...that pH have to pass through which value? it is the pkR value which is 6. and then you can know that the pH have to pass though pkA of 10 for the 0 to -1 change to occur. Notice the trend, as you increase the pH, you are taking H+ away from the molecules, whichever functional group that have the lowest pkA will lose the H+ first, the highest pkA will lose it last. ect...to calculate the pI, you only concern yourself with pkA associated with the -1 to 0, and the 0 to +1 change, which is what "average the two pKa values on either side of the neutral form of histidine" mean. You can make it easier to see by putting the pkA value between each structure from lowest to highest (you ahve 4 structures so that leave 3 spaces between them= 3 pkA value).

#### docntrainin

You should know that the pkA of all COOH is 2 and NH3 is 10 for all amino acids. so you can deduct that the other pka is the pkR. (pkA of the side chain)
taht being said the picture on that website is lacking the labelling for pkA. The principle is simple, lets say when the overall charge is +2, as it is changing from being +2 to +1, the pH have to be higher than a certain value for the OH to become O-, that value is the pkA of COOH group, which is 2 (you can see why if you review acid-base equilibrium and definition of pka). you can use this to find out that from +1 to 0, it is the R side chain (NH+) becoming NH, that cause the change in overall charge. So...that pH have to pass through which value? it is the pkR value which is 6. and then you can know that the pH have to pass though pkA of 10 for the 0 to -1 change to occur. Notice the trend, as you increase the pH, you are taking H+ away from the molecules, whichever functional group that have the lowest pkA will lose the H+ first, the highest pkA will lose it last. ect...to calculate the pI, you only concern yourself with pkA associated with the -1 to 0, and the 0 to +1 change, which is what "average the two pKa values on either side of the neutral form of histidine" mean. You can make it easier to see by putting the pkA value between each structure from lowest to highest (you ahve 4 structures so that leave 3 spaces between them= 3 pkA value).
Thank you thank you thank you! This makes so much more sense now. Just one last question, when you are drawing your diagrams, do you always work yourself up to a higher pH i.e. start with the lowest pH and then continue to raise it and draw the diagram accordingly.

#### tn4596

7+ Year Member
Thank you thank you thank you! This makes so much more sense now. Just one last question, when you are drawing your diagrams, do you always work yourself up to a higher pH i.e. start with the lowest pH and then continue to raise it and draw the diagram accordingly.
you can do it whichever way you feel comformtable with, just make sure the pkA and charges varies accordingly with your pH level
ie. if you start with high pH and work toward lower pH, you are adding more H+ to the structure as you go from left to right, the charge goes from negative to positive, and pkA is moving from high to low.

#### geeyouknit

7+ Year Member
What is the isoelectric point for cysteine?

Information given in table:

pKa COOH (1.9) pKa NH3 (10.3) pKa R group (8.4).

Answer: 5.2. The isoelectric point is found by averaging the pKas that give a +1 charge to the molecule and a -1 charge to the molecule. Thus from the table values, pI = 1.9+8.4/2 = 10.3/2 = 5.15.

I understand that the isoelectric point is where zwitter ion exist and that it is where the +1 -1 charge is aka a neutral molecule, but how did they know which pKas to use? I just averaged the pKas of COOH and NH3. How did they know to use pKa of R group and pKa of COOH.

So confused! Thank you
At a pH of 5.2 (the pI), wouldn't there be a charge of +1 ? The COOH would be -1, and both NH3 and R group would have + 1 ( +2)

Thanks.

Edit: Ok, I think I get why. The R group is a thiol which is more likely to deprotonate than protonate (more like an acid than base).
But if you didn't know it was cysteine or didn't know it was thiol, how could you tell from just "R" group?

Last edited:

#### tn4596

7+ Year Member
you cant really tell the charge if you don't know the identities of the amino acids or the side chains..on these quetions they will probably even give you the structure of the amino acid on the MCAT; you don't get to memorize all the amino acids till biochem...

#### geeyouknit

7+ Year Member
you cant really tell the charge if you don't know the identities of the amino acids or the side chains..on these quetions they will probably even give you the structure of the amino acid on the MCAT; you don't get to memorize all the amino acids till biochem...
Right. I'm saying that if they told me it was thiol, I would know the charge.
But if they just said R group with a pH, I can't say for sure if its an acid or base.
In the OP's question, they just said cysteine. I'm guessing they want you know cysteine has a thiol group? Not sure how else you would know.

#### MShopes

5+ Year Member
What is the isoelectric point for cysteine?

Information given in table:

pKa COOH (1.9) pKa NH3 (10.3) pKa R group (8.4).

Answer: 5.2. The isoelectric point is found by averaging the pKas that give a +1 charge to the molecule and a -1 charge to the molecule. Thus from the table values, pI = 1.9+8.4/2 = 10.3/2 = 5.15.

I understand that the isoelectric point is where zwitter ion exist and that it is where the +1 -1 charge is aka a neutral molecule, but how did they know which pKas to use? I just averaged the pKas of COOH and NH3. How did they know to use pKa of R group and pKa of COOH.

So confused! Thank you
I think it is not that hard. First, you consider the overall charge of the molecular by generally looking at how many NH3+ are there in the first place. Say the overall charge is +1, you would then use the first Pka value to get the neutral molecule which is usually by deportonating the COOH group (- charge . Once you see the molecular is neutral, use that Pka and the next Pka to calculate the PI. The reason is because the PI would be in the middle and still would be considered the same structure as the lowest pka in your calculation (which is the natural form). If you are good at it, I think you can do it all in your head.

For cysteine, they should give you the structure and you should see that there is one NH3 group and one COOH and one SH. Luckily, they specify which Pka is for which group. From looking at it, the molecular has a +1 charge...so once you deportonate the COOH, you get COO- and a neutral molecular (- +). Use that Pka of COOH that lead you to a neutral molecular and the next Pka right away which is here 8.4 to get you the answer.