Peptide Net Charge

[gea113]

Hi,

I came across this question today about calculating the net charge on a peptide at pH 7, and I don't get where I'm going wrong.

The peptides given were:

A) AVDEKMSTRGHKNPG
B) YPGRSMHEWDIKAQP
C) HIPAGEATEKALRGD
D) EAPDTSEGDLIPEUS

According the answer, only C and D have a negative net charge. C has a negative net charge of -1, while D has a net charge of -5. Using the rule that basic amino acids (histidine, lysine, and arginine) provide a +1 charge, while acidic amino acids (glutamate and aspartate) provide a -1 charge, I thought the answer would be:

A) -2
B) -1
C) 0
D) -5

What am I missing here??
Thank you!

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[aldol16]

Hi,

I came across this question today about calculating the net charge on a peptide at pH 7, and I don't get where I'm going wrong.

The peptides given were:

A) AVDEKMSTRGHKNPG
B) YPGRSMHEWDIKAQP
C) HIPAGEATEKALRGD
D) EAPDTSEGDLIPEUS

According the answer, only C and D have a negative net charge. C has a negative net charge of -1, while D has a net charge of -5. Using the rule that basic amino acids (histidine, lysine, and arginine) provide a +1 charge, while acidic amino acids (glutamate and aspartate) provide a -1 charge, I thought the answer would be:

A) -2
B) -1
C) 0
D) -5

What am I missing here??
Thank you!

How are you doing your math? Showing your work will allow you to find your error.

A) There are two negatively-charged amino acids and four positively charged ones, giving it a net +2 charge.

B) There are three positively-charged amino acids and two negatively-charged ones, giving it a net charge of +1.

C) There are three positively-charged amino acids and three negatively-charged ones for a net charge of 0. Not sure where they get -1. Perhaps they count histidine as neutral since its pKa is so close to 7 so that a non-negligible portion of it will still be deprotonated at pH 7.

D) There are five negatively-charged amino acids and no positively-charged ones, giving you a net charge of -5.

Now see if you can find the individual amino acids. Are you familiar with the one-letter naming system?

 

[Lawpy]

Hi,

I came across this question today about calculating the net charge on a peptide at pH 7, and I don't get where I'm going wrong.

The peptides given were:

A) AVDEKMSTRGHKNPG
B) YPGRSMHEWDIKAQP
C) HIPAGEATEKALRGD
D) EAPDTSEGDLIPEUS

According the answer, only C and D have a negative net charge. C has a negative net charge of -1, while D has a net charge of -5. Using the rule that basic amino acids (histidine, lysine, and arginine) provide a +1 charge, while acidic amino acids (glutamate and aspartate) provide a -1 charge, I thought the answer would be:

A) -2
B) -1
C) 0
D) -5

What am I missing here??
Thank you!

At pH = 7, D and E have -1 charge and H, K and R have +1 charge. The rest of amino acids have 0 charge, -COO and -NH3 endings of the peptide cancel each other, so side chain charges contribute to overall peptide charge.

A) AVDEKMSTRGHKNPG --> D, E, K, R, H, K --> 2*(-1) + 4*(+1) = +2.

B) YPGRSMHEWDIKAQP --> R, H, E, D, K --> 3*(+1) + 2*(-1) = +1.

C) HIPAGEATEKALRGD --> H, E, E, K, R, D --> 3*(+1) + 3*(-1) = 0.

D) EAPDTSEGDLIPEUS --> E, D, E, D, E --> 5*(-1) = -5

So only peptide D has a negative charge

 

[Lawpy]

C) There are three positively-charged amino acids and three negatively-charged ones for a net charge of 0. Not sure where they get -1. Perhaps they count histidine as neutral since its pKa is so close to 7 so that a non-negligible portion of it will still be deprotonated at pH 7.

But that means H = 0 for A) and B). I don't know what the correct answers are but I could see it being the case. Take a look at this chart:

Unlike arginine and lysine, histidine side chain pKa is above physiological pH and yet it is viewed to be basic. I think local acidic conditions contribute to histidine being basic due to its buffering property useful in enzymatic reactions. It's weird because from the Henderson-Hasselbalch equation:

pH = pKa + log ([base]/[acid]) -->
pH = pKa + log ([deprotonated]/[protonated]) -->
7 = 6 + log([deprotonated]/[protonated]) -->
1 = log ([deprotonated]/[protonated]) -->
[deprotonated]/[protonated] = 10

This means at pH = 7, ~91% of histidine exists in deprotonated form and yet it's considered basic in physiological conditions.

 

[aldol16]

Unlike arginine and lysine, histidine side chain pKa is above physiological pH and yet it is viewed to be basic. I think local acidic conditions contribute to histidine being basic due to its buffering property useful in enzymatic reactions. It's weird because from the Henderson-Hasselbalch equation:

You mean below physiological pH. The pKa of histidine is 6 whereas physiological pH is 7. Therefore, it's actually relatively acidic compared to physiological pH. It's also not weird - it's actually very very convenient once you begin to understand advanced biochemistry. The pKa also varies depending on the local environment. Remember that these pKas are all measured in the free amino acid form whereas amino acids are not found floating around freely in the cell. In a protein, if a His residue is buried, it's more likely to have a lower pKa (more acidic) because it wants to get rid of the charge - unless there's an ion it can pair with around, in which case it would have a higher pKa than the free form.

This means at pH = 7, ~91% of histidine exists in deprotonated form and yet it's considered basic in physiological conditions.

It's considered basic under physiological conditions mainly because the interesting chemistry it does involves it acting like a base (and as an acid). This is because if you have something that's strongly basic, once you give it a proton, it doesn't want to give it back. That's why you don't usually see Lys or Arg in active site chemistry unless there are other ion-pairing or H-bonding effects going on so that their pKas are altered. But His is different because it doesn't really care if it has a proton or not - you can give it a proton to store for a bit and it'll give it right back without hassle.

 

[Lawpy]

You mean below physiological pH. The pKa of histidine is 6 whereas physiological pH is 7. Therefore, it's actually relatively acidic compared to physiological pH. It's also not weird - it's actually very very convenient once you begin to understand advanced biochemistry. The pKa also varies depending on the local environment. Remember that these pKas are all measured in the free amino acid form whereas amino acids are not found floating around freely in the cell. In a protein, if a His residue is buried, it's more likely to have a lower pKa (more acidic) because it wants to get rid of the charge - unless there's an ion it can pair with around, in which case it would have a higher pKa than the free form.



It's considered basic under physiological conditions mainly because the interesting chemistry it does involves it acting like a base (and as an acid). This is because if you have something that's strongly basic, once you give it a proton, it doesn't want to give it back. That's why you don't usually see Lys or Arg in active site chemistry unless there are other ion-pairing or H-bonding effects going on so that their pKas are altered. But His is different because it doesn't really care if it has a proton or not - you can give it a proton to store for a bit and it'll give it right back without hassle.

My bad I meant below. Yeah local acidic conditions are involved in making histidine a useful amino acid for catalytic reactions, but that's mainly due to its buffering and amphoteric properties. The chemistry of side chains would make histidine acidic in physiological conditions, so you would expect histidine to have a 0 charge, not +1. And yet textbooks classify histidine as a basic amino acid simply because it can accept a proton. This is misleading for questions that ask about net charge of a peptide at pH = 7, because histidine here does in fact have 0 charge, not +1.

 

[aldol16]

My bad I meant below. Yeah local acidic conditions are involved in making histidine a useful amino acid for catalytic reactions, but that's mainly due to its buffering and amphoteric properties. The chemistry of side chains would make histidine acidic in physiological conditions, so you would expect histidine to have a 0 charge, not +1. And yet textbooks classify histidine as a basic amino acid simply because it can accept a proton. This is misleading for questions that ask about net charge of a peptide at pH = 7, because histidine here does in fact have 0 charge, not +1.

Again, it's much more complicated than you're making it because under "physiological conditions," histidine is never by itself. Imidazoles bind metal ions quite readily and you don't want that happening to your labile iron pool. The only relevant histidines are the ones in proteins and their pKas will vary based on local environment. Hence why you can't say whether it will be positive, neutral, or have significant populations of both. In this case, it's a peptide and we don't know the local structure of the peptide. Usually, we make the simplification in an introductory biochemistry course that histidine in all cases has a pKa of 6 (which is not actually true) and so if you take that to be the case, then the answer is that histidine here has both zero and +1 charge. 10% is non-trivial. I wouldn't make a fuss about this if we were talking about pH 8 where it'd be closer to 1% but the truth is, under "physiological conditions," histidine can be either mostly protonated or mostly deprotonated. It depends on the local environment.

 

[Lawpy]

Again, it's much more complicated than you're making it because under "physiological conditions," histidine is never by itself. Imidazoles bind metal ions quite readily and you don't want that happening to your labile iron pool. The only relevant histidines are the ones in proteins and their pKas will vary based on local environment. Hence why you can't say whether it will be positive, neutral, or have significant populations of both. In this case, it's a peptide and we don't know the local structure of the peptide. Usually, we make the simplification in an introductory biochemistry course that histidine in all cases has a pKa of 6 (which is not actually true) and so if you take that to be the case, then the answer is that histidine here has both zero and +1 charge. 10% is non-trivial. I wouldn't make a fuss about this if we were talking about pH 8 where it'd be closer to 1% but the truth is, under "physiological conditions," histidine can be either mostly protonated or mostly deprotonated. It depends on the local environment.

What are the local conditions where histidine in a peptide will have higher pKa than free histidine? You said it would happen if histidine participates in an ion pair with a nearby ion. Would this work with a nearby glutamate or aspartate residue?

 

[aldol16]

What are the local conditions where histidine in a peptide will have higher pKa than free histidine? You said it would happen if histidine participates in an ion pair with a nearby ion. Would this work with a nearby glutamate or aspartate residue?

Yes it would. Ion pairing between an acidic residue and histidine would have the effect of lower the pKa of the acidic residue and raising the pKa of the basic residue so that the ion pairing is more facile. See this paper for His-Asp base pairing: http://pubs.acs.org/doi/pdf/10.1021/bi962546x. Pay special attention to Figure 3 and Table 3. The pKa of His is elevated above 6 and the pKa of Asp is lowered from 3.9.