Amino Acid pI Question

[justadream]

pkA of COOH is around 2
pKa of NH2 is around 9.5

Let's assume we look at an amino acid with a non-fancy (that means, like alanine) side-chain.

The pI is around 5.75.

If the pH of the environment = pI, the amino acid exists as a zwitterion (NH3+, COO-).

What about if the pH is slightly greater than the pI? In this example, if the pH is 6, then what happens?

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[Czarcasm]

pkA of COOH is around 2
pKa of NH2 is around 9.5

Let's assume we look at an amino acid with a non-fancy (that means, like alanine) side-chain.

The pI is around 5.75.

If the pH of the environment = pI, the amino acid exists as a zwitterion (NH3+, COO-).

What about if the pH is slightly greater than the pI? In this example, if the pH is 6, then what happens?

I always forget this stuff, but reasoning it out helps me remember. What happens to any acid-base pair when the pH increases? Well, a pH increase means a more basic solution. So if at pH=pI we have a zwitteron (NH3+ and COO-). If we increase the basicity, that is, we increase the pH above pI (particularly, above the pKa of the NH3+), then the NH3+ will deprotonate to NH2 (an uncharged species), and the overall charge will be (-1 + 0 = -1). If we decrease the pH below pI (particularly, below the pKa of the COO-), a more acidic solution, then the COO- will be protonated to COOH and the overall charge will be (0 + 1=+1). It gets a little more tricky when you have an R group with a pKa. This was a simple scenario of a non-polar aminoacid and so we only needed to consider the pKa's of the terminal amino and carboxyl groups - but that's a scenario less presented on the MCAT.

 

[justadream]

@Czarcasm

I originally was using that logic but think about this:

What if you were just considering a NH2 alone (pKa = 9). Would you agree that at a pH of 6, it should still be protonated (NH3+ form as opposed to NH2)?

After all, the pH of 6 would still be less than the pH of the NH2.

Why, when you are consider amino acids, does the pI matter? Why don't you just consider the pKas of the NH2 and COOH separately?

 

[Czarcasm]

@Czarcasm

I originally was using that logic but think about this:

What if you were just considering a NH2 alone (pKa = 9). Would you agree that at a pH of 6, it should still be protonated (NH3+ form as opposed to NH2)?

After all, the pH of 6 would still be less than the pH of the NH2.

Why, when you are consider amino acids, does the pI matter? Why don't you just consider the pKas of the NH2 and COOH separately?

Right. If the pH is 6 and since the pKa of the amino terminus is 9, then we'd have 1000:1 times more of the NH3+ form over the NH2 form, which for all intents and purposes is 100% NH3+. We relate the carboxyl group in a similar way to pH, keeping in mind the log-based relationship and HH equation. Finding the pI is useful because it tells us when the species on average is in it's neutral form (uncharged). I'm not really to keen with biotechniques, but this is particularly useful for separating different compounds. I vaguely recall one particular approach (I think it's 2D electrophoresis), where different compounds are first separated by the electric field along a pH gradient until no charge exists and then they are separated again by size, which allows for great resolution and isolation for studying different proteins and structures. Anyways, I don't think that's too important here. It's something I recall from biochemistry a while ago. For the sake of the MCAT though, I'd just understand how to find the pI and understand what it means and how it can change relative to the pH of the solution.

 

[justadream]

@Czarcasm

Can you explain the "the species on average is in it's neutral form"?

I guess you are saying that at the pI, that's when you have the most species in the neutral form.

But at the pI, can you conclude how much (roughly) is really in the neutral form? Is it like over 50%?

 

[Czarcasm]

@Czarcasm

Can you explain the "the species on average is in it's neutral form"?

I guess you are saying that at the pI, that's when you have the most species in the neutral form.

But at the pI, can you conclude how much (roughly) is really in the neutral form? Is it like over 50%?

There are different variations of the HH equation. All essentially tell us the same thing, but the one I prefer is this:

HH = pKa + log([Base]/[Acid]) because it's the one that makes the most conceptual sense to me and allows for easy approximation. Consider your example of alanine, which has 2 pKa's: the carboxyl terminus (approx 3) and the amino terminus (approx 9). If for instance, this aminoacid is in a solution of pH=6, what this means is that (based on the HH equation), we have roughly 1000 times more of NH3+ (the acid form) than NH2 (the base). Why? Because we are 3 pH units lower than it's pKa (the equivalent of 1000:1). Likewise, for the carboxyl terminus, we'd have roughly 1000 times more of COO- (the base form) since we are 3 pH units higher than it's pKa. So all in all, on average, a given amino acid has a positive end (NH3+) and a (COO-) end. However, it's possible to have a species that has a neutral NH2 end and even a COOH end at this pH. It's uncommon, but not impossible. Regardless, we focus on the zwitterion, the neutral species, which is also the major species when pH=pI. Deviating away from this pH will change the ratios and you may have a solution of aminoacids that is either more positively charged or negatively charged on average, depending on which way you shift the pH.

 

[texan2414]

What do you mean by "at pI how much roughly is the neutral form"?
From what I remember, at pI, the total net charge on the peptide is zero. There is no proton half-way titrated.

Also, pH and pI are important because they can affect solubility of a protein in an aqueous solution.

 

[justadream]

@texan2414

Well when the pH = pI, that's the environment in which you maximize the # of amino acids that exist as a zwitterion (that is, NH3+ and COO-).

However, not ALL of the amino acids will exist as a zwitterion. Some might be positively charged (NH3+, COOH) and some might be negative (NH2, COO-). Perhaps the vast majority will be in the zwitterion form but I was asking if you could quantify/estimate this number.

 

[texan2414]

I see.

 

[Shirafune]

This is how my biochem professor taught it.

1) Write down the different charged states the amino acid can have.
2) Starting with the highest positive charge, write them from left to right.
3) Assign pKas in ascending order in order of deprotonation. (you hopefully won't need to memorize sidechain pKas)

Take lysine for example. It is a basic amino acid, so it should have two amino groups and one carboxylic acid group.

1,2) Therefore, it charges go from +2 -> +1 -> 0 -> -1
3) The order of pKas is 2, 9.5, 10.5 (carboxylic group, amino group, side chain)
4) Use the Henderson-Hasselbalch equation to figure out the ratios of the amino acid's different forms.