Biochemistry from A to Z! Remember, amino acids are amphiprotic, amphoteric, and zwitterionic*! (at neutral & bodily pH). Amino acids are protein letters (and some also moonlight in a bunch of other roles in our body) and when they’re in their free form (not linked up into chains) they can be described by these weird terms are words we can use to describe molecules that kinda have split personalities. Amphiprotic & amphoteric describe how the molecules can act in 2 “opposite ways” (acting as an acid OR a base); and zwitterionic describes how a neutral molecule can have fully positive and fully negative parts. Let’s look in some more detail – starting with my fave…

first – uber quick review: Molecules are made up of atoms (individual carbons, hydrogens, etc.) and atoms are made up of smaller parts called protons (which are positively-charged) and neutrons (neutral) that hang out together in a dense central nucleus and are surrounded by a “cloud” of negatively-charged electrons they interact with other atoms through. In order to link together, they kinda merge their clouds, sharing pairs of electrons to form strong, covalent, bonds and they don’t always share fairly, which can lead to partial charge imbalance. The # of protons defines an element (e.g. carbon always as 6 and hydrogen always has 1). But the # of electrons can change (which is how you get fully charged particles (ions) & the # of neutrons can also change (which is how you can get radioactive isotopes).  We can refer to molecular charge (# protons – # electrons in the entire molecule) and atomic charge (# protons – # electrons “owned” by an atom within a molecule) and therefore molecules that are neutral overall can have fully and partially charged parts, as is the case with zwitterions

zwitterion: something that is overall neutral, but has fully positive & fully negative parts. 

The name comes from a German prefix meaning “hybrid” – and, as discussed above, an ion is a charged molecule. We call something an “anion” if it has a negative charge (which comes from having more electrons than protons) and we call something a “cation” if it has a positive charge (comes from having more protons than electrons). And a zwitterion is a single molecule that is both! It has both negative and positively-charged parts, yet is neutral overall. (though, as we’ll see, zwitterionicness is pH-dependent so something can be a zwitterion at one pH but not at another pH). 

The classic example of a zwitterion is the amino acid. At physiological (bodily) pH (~7.4), its amine group is protonated and cationic (-NH₃⁺) and its carboxylic acid group is deprotonated and thus anionic (-(C=O)-O⁻). Those charges cancel out, so the molecule is neutral overall. But it has those full, separated and opposite charges. 

note: Do not confuse “zwitterionic” with “polar” which is a broader term which refers to molecules with a separation of charge. Sound similar, right? Difference is, with polar molecules you can be talking about partial charges (such as in water how the oxygen is partly negative because it hogs electrons from the hydrogens and the hydrogens are therefore partly positive). And, with polar, the molecule can be neutral or charged overall. With a zwitterion, atoms within the molecule are assigned full, “formal” charges meaning that those atoms have an unequal number of protons and electrons, not merely “less electron density” coming from a shared bond. 

As I mentioned, zwitterionic is kinda like a “mood” that a molecule can be in. Just like your mood might depend on your environment, a molecule’s charged state depends on its environment. So let’s look closer at where those charges on the amino acid are coming from….

First off, what is an amino acid anyway? Like many (but not all) things in science, hints are in the name. At the core is the central (alpha) carbon (Cα) – which is hooked up to the unique charm (side chain or “R group”) which gives different amino acids their special “superpowers” and the generic parts that allow for linking and make it an amino acid – “Amino” refers to them having an “amine” group – a nitrogen (N) hooked up to hydrogen(s) (H) and/or carbon(s) (C). And “acid” refers to them having a carboxylic acid group – a C double-bonded to an oxygen (O) and also bonded to a hydroxyl (-OH) group (so (-(C=O)-OH). An “acid” (in one definition) is something that donates a proton (an H⁺), and a carboxylic acid can donate a proton from the hydroxyl group to give you a carboxylate anion (-(C=O)-O⁻). The neutral form of the amine group (-NH₂) can act as a base (proton taker) to become the cationic -NH₃⁺. ⠀

Which protonation state these end groups are in depends on the pH (a measure of how many free protons are floating around). As mentioned above, at physiological pH the “zwitterionic” form is the most common. But, if you were to go to a really low pH (very acidic solution, lots of protons around) the carboxylate group would protonate to give the carboxylic acid form. That “un-negativizes” the carboxylate, but you still have the protonated amine group. So now the amino acid is positively-charged! And thus, by definition, it can’t be a zwitterion! If you were to start at neutral & go the other way, removing protons from the solution, raising the pH to a more basic/alkaline condition, now that amino group will deprotonate, “un-positivizing” it. And the carboxylate group is also deprotonated, and thus negative, so now the molecule is negative overall. And thus not zwitterionic! So, “zwitterionic” is a pH-dependent form a molecule can take, not a permanent state of being. But, under most conditions we talk about in biochemistry, free amino acids* will be in their neutral, “isoelectric” form**

* note: amino acids are the “free-floating” forms of protein letters – when they link together, they do so by joining the carbonyl (C=O) carbon of one amino acid to the nitrogen of the amino group of the other amino acid, physically losing the equivalent of water (2 H & 1 O)  in the process – and also “functionally” losing the free amino and carboxyl groups that are now merged into a peptide bond. As a result, when it comes to the generic peptide backbone, only the “first” amino acid will have a free amino group (we call this the “N terminus” and only the “last” amino acid will have a free carboxylic acid group (we call this the “C terminus”) 

** note: this refers to amino acids who have neutral side chains. Some amino acids have “basic” or “acidic” side chains that can also give and take protons and therefore they have more complicated isoelectric points. 

How do we know this? We can figure out what charged state of each “potentially-charged group” will predominate at a particular pH by looking to those groups’ pKa’s. Much more on this in recent posts, but key thing to know is that pKa is a measure of an acid’s strength. The lower the pKa, the stronger the acid (more easily it gives up a proton to become its conjugate base). And the higher the pKa, the weaker the acid (and thus stronger its conjugate base). 

Numbers-wise, the pKa of an acid refers to the pH at which half of the copies of that acid are deprotonated and half are protonated. And pH is a measure is a measure of free proton concentration ( [H⁺] ) in a solution. It’s an inverse log, so the higher the proton concentration, the lower the pH (more acidic the solution) and the lower the proton concentration, the higher the pH (more basic/alkaline the solution).

The stronger the acid, the more likely it is to deprotonate and stay deprotonated, even if the solution tries to bribe it with a bunch of free protons (i.e. even at a low pH). Therefore, stronger acids, will have lower pKa’s, not protonating unless the solution is at a super low pH. 

The pKa of the carboxylic acid group of free amino acids (we often refer to this as pKa1) is ~2-2.4.

Therefore, at physiological pH, it will almost all be in the deprotonated, negatively-charged form.

Note that I gave a range for pKa. The exact pKa will depend on the influence of the R group (even if that R group is neutral it still slightly alters the local environment). 

How about bases? A base is really just the flip side of an acid – because once something acts as an acid and donates a proton, it can then take back a proton (act as a base). We can therefore refer to conjugate acid/conjugate base forms of a single “acid” and use the same pKa term to look at the strength of bases based on the strength of its conjugate acid. In this case, the higher the pKa, the stronger the base (because the weaker the conjugate acid). A stronger base will more readily take and hold onto protons, even if there aren’t many available (i.e. even in a basic/alkaline solution). Therefore, it will be protonated even at high-ish pHs. 

The pKa of the amino group of free amino acids (commonly referred to as is pKa2) is ~9-10.5.

Therefore, at physiological pH, it will almost all be in the protonated, positively-charged form.

If molecules have multiple “potentially-charged groups” we can determine a value called the isoelectric point (pI) which is the pH at which the molecule is neutral overall. 

When you have a simple amino acid with a neutral side chain, this pI is simply the average of the 2 pKa’s (pI = (pK1 + pK2)/2). But with amino acids whose side chains have their own pKa’s, things get more complicated. 

You can find a chart of the various pKa values for free amino acids here: 

But then you link them up to form proteins and… It can get really hard to calculate the pI of proteins, but there are websites like ProtParam that can do it for you.   But note that they can only give you an estimate because the true pKa of any amino acid acidic group is context-dependent. 

Now let’s go into those other terms I mentioned – amphiprotic & amphoteric. The difference between them is subtle. Both refer to molecules that can act as *both* an acid and a base (amphi- means “both”). But they use different definitions of acid and base…

Note: this distinction has basically never come up in my work so you might need to know it for school, but otherwise I wouldn’t worry much about it. Just know that, as long as something is acting as an acid and a base (in any definition) you can call it amphoteric and be safe! You’ll almost always just see amphoteric used, and you call an amphoteric molecule an ampholyte. More on how this comes up later, but first let’s get back to those definitions. 

The “acid” and “base” behavior I’ve been talking about above (proton giving/taking) is the Bronsted-Lowry (BL) way of defining acids & bases. There’s also the Lewis definition, which is in terms of electron pair givers (Lewis bases) & takers (Lewis acids). More on this here: 

Acids, according to Bronsted, are things that donate protons and bases are things that accept them. Turns out a guy named Lewis didn’t quite agree regarding what the definitions of acid & base should be! In Lewis’ mind, an acid is something that accepts a pair of electrons and a base is something that donates a pair of electrons. The Lewis definition makes sense in terms of talking about nucleophiles (things that want to donate electrons) & electrophiles (things that want to accept electrons). Lewis bases donate electron pairs and so do nucleophiles. And Lewis acids accept electron pairs, and so do electrophiles. So electrophiles are Lewis acids. And “acids” and “bases” in the Bronsted sense are just a special case where the electrophile involved is a proton (H⁺). 

So, all BL acids and bases (proton giver/takers) are also Lewis acids and bases. BUT, not all Lewis acids and bases (electron pair taker/givers) are BL acids and bases. Most of the time in biochemistry, we deal with BL acids & bases, but the Lewis-only kind do show up. Especially when talking about things involving metals. Metals have big, disperse, electron clouds that are often positively charged and thus don’t mind if some electrons sneak in… thus they commonly act as Lewis acids, accepting a pair of electrons from another molecule to form coordinate complexes. 

But back to the amphiprotic vs amphoteric distinction…

As the “protic” hints at, amphiprotic refers to molecules that can act as both an acid and a base of the Bronsted-Lowry definition kind – they can both give and take protons. 

Amphoteric is more all-encompassing. It refers to molecules that can act as both an acid and a base of “any” kind – so, even those of the Lewis-only definition. 

Thus, if amphiprotic, also amphoteric. BUT

If amphoteric, not necessarily amphoteric

Let’s look at some examples. 

amphiprotic: can act as both Bronsted-Lowry acid & bases, can give and take protons

We spent several paragraphs earlier talking about a key example, amino acids! The amino group can act as a base, the carboxylic acid group can act as an acid and voila! You’ve got yourself an amphiprotic (and amphoteric) molecule. 

Other common examples…


H₂O + H₂O ⇌ H₃O⁺ + OH⁻ 

You can see that one of those waters accepted a proton and one of them donated a proton. Water is thus acting as both an acid & a base (in the BL-sense).


if it acts as a Bronsted-Lowry acid (proton donor)…

HSO₄⁻ + H⁺  ⇌ H₂SO₄

if it acts as a Bronsted-Lowry base (proton acceptor)…

HSO₄⁻ + OH⁻ ⇌ SO₄²⁻ + H₂O

If something is amphiprotic it is also amphoteric, but determining if something is “only” amphoteric is trickier… Thankfully it doesn’t come into play very frequently in biochemistry! One place you might see it is with metal hydroxides, which can often act as Lewis acids even though they don’t have protons to donate. 

For example, take aluminum oxide (Al(OH₃)). it can act as a base in the BL-way when mixed with acids – for example

Al(OH₃) + 3 H⁺  ⇌ Al³⁺+ 3 H₂O 

here you can see the hydroxide accepting protons to form water

but when it reacts with a base things can get weird…

Al(OH₃) + OH⁻ ⇌ [Al(OH₄)]⁻

The aluminum ions are accepting a pair of electrons from the oxygen, letting it join into the complex aluminate ion. Since it accepted a pair of electrons, it’s acting as a Lewis acid. But it didn’t donate any protons, so it’s NOT acting as a BL-acid. 

If you want to know more, chemguide has great info: 

Final note: As I mentioned, amphoteric molecules are called ampholytes. You might hear the term “ampholytes” in the context of isoelectric focusing (IEF), which is where you separate molecules (often proteins) based on their isoelectric point (pI) by sending them through a pH gradient towards charged electrodes. The molecules will only be compelled towards an electrode when they have an opposite charge. They’ll start traveling through it but the pH will be changing as they do. And at some point they’ll reach a point at which the pH = their pI and they’ll be neutral and stop moving. If desired, you can then separate the proteins further by taking that IEF strip, and placing it horizontally on top of an SDS-PAGE gel to separate the proteins by size. This will give you 2D electrophoretic separation which can be good for separating complex mixtures of proteins. 

A mix of “carrier ampholytes” is used to set up that pH gradient. You send them through a gel with an electric field (before your proteins) and they’ll sort themselves out based on their pI, establishing that pH gradient you’ll need. Depending on the pI’s of the things you’re trying to separate you can get mixes of these ampholytes with different pIs which will give you different pH gradients. 

So, there you have it – a small slice of biochemistry, A to Z! (or I guess Z to A…)

more on pH & pKa: 

more on protein charge:

more about all sorts of things:  #365DaysOfScience All (with topics listed) 👉 or search blog:              

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