Aw, shoot – it’s time to troubleshoot (again) because this prickly protein doesn’t like to elute! Good thing I’m not charged by the hour for my research because I’ve spent lots of hours these past couple days trying to exploit my PROTEIN’S CHARGE to purify it using ION EXCHANGE CHROMATOGRAPHY – but this protein likes to show me that it’s the one in charge! (but is it positively-charged (cationic) or negatively charged (anionic)?). Do we need to call a pI to investigate a potential case of an isoelectric point “mispoint?”
I’ve been having trouble purifying this one protein, so I was Googling for advice and I came across this article which seemed like the biochemist’s version of something like “Raising a Spirited Child” – “Purifying Challenging Proteins” – and to be honest I think this is pretty unfair to the protein! It wasn’t “challenging” when it was doing its own thing, it just doesn’t like being yanked out of its natural home and put in a weird new environment where you keep making it stick and unstick to stuff
One of a grad student’s biggest aids is Google! Seriously – you can find some great information and scientists are often asking questions and getting advice from other scientists (of course those scientists can be wrong, so it’s always important to remember to use those critical thinking skills to think about whether or not what you read makes scientific sense! which is also one of the reasons I’m so passionate about teaching people how methods work at the molecular level!).
But anyways, this morning I was Googling for advice on ION EXCHANGE CHROMATOGRAPHY – which is a protein purification technique in which you separate proteins by their charge by getting them to bind to oppositely-charged resin (little beads) in a column, washing the other stuff off, and then pushing them off with salt – the more opposite the protein & bead charges are, the stronger the charge-charge interactions, so the more salt it’ll take to get pushed off. So if you gradually increase the salt you can push proteins off based on their charge, separating them in the process.
If your protein’s +-charged (we call this cationic) you’ll want – charged resin (like an S column) but if your protein’s -charged (we call this anionic) you’ll want + charged resin (like a Q column). So how do you know what to use? You turn to a pI predictor – but they’re only a “best guess”!
Is your protein proton-greedy? Do you want to find out? Call up a pI to see what they’ll charge! Instead of “private investigator” pI in the protein investigator world stands for the ISOELECTRIC POINT, and it is the pH at which a protein is charge-neutral (overall). And understanding *why* is a great way to explore WEAK ACIDS & BASES in general. So I hope this post on isoelectric point doesn’t disappoint!
If you think about acids and bases as “cookie monsters” donating (in the case of acids) or taking (in the case of bases) proton “cookies” from a “proton cookie jar,” proteins are like a big linked-up group of friends where some of them are cookie monsters. Protons are positively-charged, so giving & taking them alters the protein’s charge. How many of these friends a protein has and how greedy or generous they are under their current conditions will determine how charged the protein is under those conditions. And we can take advantage of this charge for things like ION EXCHANGE CHROMATOGRAPHY which separates proteins by charge by getting them to bind oppositely-charged resin.
If you need a review of pH, check out: http://bit.ly/2KPzBCn
But here’s the gist: Even though they’re attached by covalent bonds, bonds to H are weaker than bonds to other atoms. H’s are really small & don’t offer much latchbility so they’re relatively easily removed. Just how easily depends on what they’re attached to. Hydrogen atoms only have 1 proton & 1 electron & they often leave that electron behind when they go, so all they’re left with is a H⁺, so we often call H⁺ a proton.
These protons can come from water itself, which is MOSTLY H2O, but also OH- & H⁺. Or they can come from other molecules, including proteins. Protons can easily find a water to latch onto (forming hydronium ion (H3O+) because the vast vast majority of water is in water form (not ionized). The ions are spread out throughout the solution, but for the sake of visualization you can think of all the protons as being in a jar, and for the sake of fun analogy-ness you can think of these protons as cookies! So, water is just cookies waiting to be made!
pH is a measurement of “how full” the cookie jar is. It’s on a negative log scale, so MORE H⁺ means LOWER pH & vice versa. When there are lots of H⁺ (pH < 7) we call the solution ACIDIC & when there aren’t many (pH >7) we call it BASIC or ALKALINE. The pH is a property of the solution that comes from the properties of the things in it, which can include proton-donors (acids) and proton-takers (bases).
Back to our protons as cookies analogy – Say you have a partially-filled cookie jar and you invite over some friends. How full will that cookie jar be at the end of the night? Depends on what type of friends you invite!
A lot of the friends don’t care whether or not there are cookies in the jar. They don’t have any cookies to give and they don’t have any desire to take any. These are like “always neutral” molecules.
Some of the friends come with cookie(s) (have the potential to act as acids). But their “generosity” can vary. Some friends really want to give away those cookies, regardless of how full the jar is. These are like strong acids, which deprotonate fully when you dissolve them in water. The 6 strong acids are hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, and chloric acid.
Other friends would rather keep the cookies for themselves but they also want to be a good friend so they keep an eye on the jar and if it gets too low they’ll pitch in. These are like weak acids. Some common weak acids are sulfurous acid (H2SO3) & phosphoric acid (H3PO4).
Some acids are multiprotic – this means they come with more than one cookie they can share – often times they’ll give up the first most willingly then have a harder time giving up the other(s)
Then you have the opposite type of friends – those that come cookie-less (or at least cookie-deficient). Some of the friends are super greedy so they’ll take even if the jar’s really low. These are like strong bases, such as sodium hydroxide & potassium hydroxide.
But others, even though they want a cookie, don’t want to be too greedy so they’ll only take if there’s enough for everyone else that wants them. These are like weak bases like ammonia & sodium bicarbonate.
When an acid gives up a proton it has the potential to take back a proton – so a deprotonated acid is a CONJUGATE BASE and the opposite is true for bases – a protonated base is a CONJUGATE ACID. It has the *potential* to go back to how it was, but it might not want to because they’re happier having or not having it.
When a strong acid gives up a proton, it has no desire to take one back and when a strong base takes a proton it has no desire to to give it back. So these are essentially “irreversible.” But the weak acid was kinda iffy about giving up that proton to begin with, so it might take one back, especially if it sees that the jar’s getting fuller. And vice versa for weak bases.
You can think of a solution kinda like a middle-school cafeteria with a cookie jar. pH is a description of how full the jar is and it is a property of the cafeteria as a whole – it comes from contributions from all the students (molecules), some of whom share cookies and some of whom take them.
All these molecules are made up of atoms (of elements like hydrogen (H), carbon (C), oxygen (O), nitrogen
(N), & sulfur (S)) joined together through covalent bonds. In any solution you have a fixed # of each type of atom – that is, you can’t do alchemy & voila suddenly have gold. You can’t change the total # of hydrogens, but you can change how they’re distributed.
The atoms in proteins are arranged into amino acid building blocks that are linked up to form proteins. You can think of the individual amino acids as “students” that link up to from large “cliques.” There are 20 (common) amino acids, & proteins can be made up of “any” # of any combination of them (e.g. you can have 15 “Janes” in one clique).
We can talk about “cookie-having” at the clique (protein) level or the friend (amino acid level). At the clique (protein) level we talk pI (ISOELECTRIC POINT) and it comes from what’s happening at the friend (amino acid) level, which we measure as pKa.
Some amino acids have more H⁺s than other amino acids. If they “look at the jar” & see that there are plenty of cookies, they say “why should I give up my cookie if there’s already cookies out there?” So they keep it. Others would like a cookie but they have better “self-control” – not until the jar’s pretty full will they take some for themselves.
Different amino acid “friends” have different levels of greediness, which we describe using a # called pKa. Before we get into the details, a key thing to remember is that pH is the INVERSE LOG of the concentration of free H⁺s [H⁺] -> this means that a HIGHER pH means FEWER H⁺ & more basic/alkaline. & a LOWER pH means MORE H⁺ & more acidic
The pKa is the pH at which 1/2 of that thing will be protonated & half won’t be. Below the pKa, there are more free H⁺ (fuller cookie jar), so the thing’s more likely to be protonated. Above the pKa there are less free H⁺ (less cookies in the jar) so the thing’s more likely to be deprotonated.
But not all things are protonatable/deprotonatable. They have to have an H for one thing & that H can’t be too tightly held. Each location that is protonatable/deprotonatable has its own pKa. So let’s look where on proteins we can find these sites.
Amino acids all have the same generic backbone that they use to link together & then they have unique “side chains” aka “side groups” aka “R groups”
The generic backbone part has an amino (-NH2) group on 1 end & a carboxyl -(C=O)-OH group on the other end. They link up amino to carboxyl in an amide aka peptide bond —NH-(C=O)— , shed a water in the process & the part that’s left we often refer to as the “residue” – so when alanine is by itself, we call it a free amino acid. But when it’s in a chain (peptide) we call it a residue.
When you have free amino acids, they all have at least 2 groups that can give or take hydrogens. The amino end can exist as -NH2 (neutral) or -NH3+ (+ charged) & the carboxyl group can exist as -(C=O)-OH (neutral) or -(C=O)-O- (- charged). So a free amino acid can be +, -, or neutral. The pKas of these groups depend on the amino acid but are typically ~10 & ~2 respectively.
Physiological pH (the pH inside your body) is about 7.4, which is above 2, so most of the carboxyl groups are protonated & thus in the negatively-charged carboxylate form. But below 10, so most of the amino ends are in the +-charged NH3+ form. So the charges cancel out – we call this a ZWITTERION
When you link amino acids together, you’re only left with 1 amino end & one carboxyl end. So you only have to take those group’s protonation state into account once. But some of the side chains in the residues in between those ends can get protonated or deprotonated as well & when we talk about pKa’s for amino acids in the context of proteins or peptides, we usually are talking about the pKa of the side chain (if there is one). Sometimes this is referred to as the pKR or pK3.
There are 2 side chains that are frequently deprotonated at physiological pH – we call these ACIDIC because they donate H⁺s – & when they do they become negatively charged & now capable of accepting H⁺s (acting as a base) so we call them “conjugate bases” It can seem kinda confusing because “acidic” residues often play important roles by acting as bases in their deprotonated form. The “acidic” refers to its NEUTRAL form being acidic.
These side chains are: Aspartic acid (Asp, D)(pKa ~3.65) gives up a H⁺ to become aspartate & Glutamic acid (Glu, E)(pKa ~4.25) – gives up a H⁺ to become glutamate. Cysteine (Cys, C) & tyrosine (Tyr, Y) can can also deprotonate to give negatively charged chains, but they do so much less readily – pKa of 8.4 for Cys & 10.5 for Tyr
There are 3 side chains that are frequently protonated at physiological pH – we call these “basic.” Lysine (Lys, K) (pKa ~ 10.28) & Arginine (Arg, R) (pKa ~13.2) are predominantly H⁺ated at cellular pH, but Histidine (His, H) (5.97) is more “iffy” (remember that pKa tells you when HALF the groups are deprotonated on average so it’s not like you hit the pKa & bam they’re all deprotonated – you have a mix. Also, an important caveat is that pKas are context-dependent so the pKa you get from a table is likely close to but not exactly the “real” pKa in the situation you’re looking at.
So this is all happening at the “friend” but what if we want to know about the clique’s “cookie-ness”? We need to take into account that clique’s unique combination of friends -> We calculate the pI (isoelectric point) is the pH at which the protein is neutral overall. It doesn’t mean that each individual amino acid is neutral, just that the non-neutral ones balance out.
So proteins with high pIs are like cookie-greedy cliques. You have to get them to see that the jar’s really low & others are desperately cookie-deficient before they’ll donate (at the net level)
The higher the pI, the more H⁺-rich the protein is likely to be -> you have to take it to a higher pH (more basic meaning less free H⁺) before it will shed H⁺s. We often refer to such proteins that are + charged at neutral pH as “basic” & they get that “basicity” because they have lots of basic residues (His, Lys, &/or Arg).
Proteins with a low pI are like generous cliques. They don’t need cookies to be happy and will only take it once they know there’s plenty for everyone!
Proteins with pI’s below neutral are “acidic” and they usually have lots of Glu’s and Asp’s.
Why do we care? In addition to being biologically important, it’s biochemically useful! If we know a protein’s charge at various pHs we can do things like get it to stick to oppositely-charged resin, wash off other stuff, then change the pH to change the charge & make it less attracted to the resin (or add salt to compete it off) so it comes off.
This is the theory behind ION EXCHANGE CHROMATOGRAPHY (aka my nightmare for the past couple days) – which has 2 “flavors” – ANION exchange and CATION EXCHANGE. Ions are charged things and basically you “exchange” ions from salts (like the Na+ or Cl- of NaCl (table salt) with protein ions. Then you can gradually increase the salt concentrations so that those salt ions outcompete the protein and you get another exchange. Or you can change the pH to change the protein’s overall charge (the lower the pH, the more free H+ for the protein to latch onto -> become more positive & vice versa)
In CATION EXCHANGE chromatography you have negatively charged resin & you’re binding & exchanging positively-charged (cationic) proteins & salt ions.
ANION EXCHANGE chromatography is the opposite – you have positively charged resin & you’re binding & exchanging negatively-charged (anionic) proteins & salt ions.
The more oppositely-charged something is, the more salt it will take to compete it off. And since different proteins have different charges, they’ll come off at different salt levels.
Normally you run anion exchange OR cation exchange – but I’m purifying this protein that is a bit unpredictable. You can use tools like the free website ExPasy ProtParam to tell you what the predicted PI is – but this is just a prediction! And sometimes it seems like it’s pure fiction!
I’m trying to purify this protein, and it has a predicted pI of ~5.8 – and my buffer is at 8.0, which is well above the pI. Higher pH means fewer protons, means less full cookie jar, so the protein will donate – and when it donates those H+ it loses positive charge. A pI of 5.8 should mean that at pH 5.8 it’ll have taken and donated to the point where it’s neutral overall. And if it donates any more it’ll start turning negative.
And when something’s negative we call it anionic. We can exchange it for other anionic things in ANION EXCHANGE CHROMATOGRAPHY – the + thing stays stationary (positively-charged groups permanently stuck to the little beads in the column) while you swap out the – thing the beads are bound to – first you exchange salt (like the cation Cl- coming from NaCl (table salt) for protein – and then you exchange that for salt again (but you have to really flood it with salt at this point in order to push it off)
Normally with ion exchange I set up an automated method where I equilibrate the column, load the sample using a sample pump that uses a tube like a straw to suck up the protein solution until it senses air, wash the column with the same salt concentration it’s already at, and then goes straight to a gradient elution – gradually increasing the salt concentration and collecting what comes out in ~1mL portions in a deep-well block.
Since the predicted pI was below my buffer pH, the first time I tried purifying this protein, I reached for the Q column, which is an anion exchange resin. But my protein didn’t bind – it just went straight through – I was able to save it but it wasn’t purer – or more concentrated – which are 2 of the main reasons for doing ion exchange! So this time I tried hooking up a Q column AND and S column (a cation exchanger) back-to-back -> this way if the protein flowed through the Q because it wasn’t actually negatively-charged, it should get trapped on the S. Then, after I loaded the sample, instead of going straight to the elution, I I could separate the 2 & run gradient elutions on them separately, increasing the salt to get whatever stuck to come off – at least in theory.
But nothing came off – either of them – I thought my protein precipitated on the column. One reason this could have happened is the initial low-saltiness – In the equilibration step I wash it with a lot of buffer at the salt concentration I want to start at (which is the salt concentration my protein’s in (which is usually diluted from the salt concentration I do the initial purification steps at – proteins often aren’t happy in low salt environments – and they’re more likely to bind non-specifically because there’s less charge-based “competition” – so I normally do the affinity chromatography steps at a higher salt concentration. But with IEX you want to start low before you can go high – because you really want to be able to get a good gradient – and you need your protein to stick – so you don’t want too much competition in the beginning – so I dilute the protein first
But the problem actually was I hadn’t diluted it enough! The salt was winning from the start – I couldn’t see it from the chromatograph because it was so dilute (but not enough) but when I diluted part of the flow-through more and re-ran it – some – but just a little – weakly bound the Q (I had to run a ton on a gel to see it and the peaks where all bumpy like it was only loosely bound). So the lower salt helped, but not quite enough – so I raised the pH so there are fewer protons around (emptier cookie jar) so the protein is more likely to donate – and thus have a lower charge. I diluted it all & now have 1.2L flowing through this little 5mL column (just the Q this time now that I know which it binds) – enjoy the swim little fellas – I need to go enjoy some rest!
When we run SDS-PAGE to separate proteins by size, we want to make sure all the proteins have a normalized charge so we “hide” their natural charges with a coat of negatively-charged SDS
in summary: Key points to keep in mind
🔹 pH is a measure of free protons (H+) which are positively charged, but pH is on a negative log scale so LOWER pH means more positively-charged protons floating around
🔹some parts of proteins can grab onto them – the more there are (lower the pH) the more likely this is -> increases charge (an make neutral side chains ➕ charged or ➖ charged side chains neutral)
🔹when pH is high, kiss those H⁺ goodbye! at high pH there aren’t many protons so this is less likely and some protein parts can actually donate them to the cause -> decreases charge (➖ side chains can’t get neutralized (they stay ➖) And ➕ side chains have to “give up” their H⁺ (they get neutralized)
🔹proteins have different combinations of parts that give & parts that take
🔹as a result, a protein has an overall charge that depends on the pH
Each protein has a specific point at which it at which the protein OVERALL is NEUTRAL – there can be ➕ & ➖ charged chains, but they perfectly cancel each other out & we call this charged but neutral condition one of my favorite words 👉 ZWITTERIONIC. The pH at which it occurs the pI or ISOELECTRIC POINT
🔹 Above the pI, the protein is ➖
🔹 Below the pI, the protein is ➕
Interactions between charged chains & water contribute to keeping proteins dissolved. & like charges repel so when protein molecules are highly charged the same say they can repel each other, so usually a protein is the LEAST soluble at its pI, but this isn’t always the case
When it comes to ion exchange chromatography – the flavors refer to the type of ion that’s being exchanged – NOT the one that the resin – so anion exchange chromatography uses + charged resin to bind – charged (anionic) proteins – and cation exchange chromatography uses – charged resin to bind + charged (cationic proteins). Of course the charge of the protein is pH dependent! The further the pH of the buffer is from the protein’s pI, the more charged the protein will be. So if you want your protein to get the protein to bind tighter, run farther away! Raise the pH for anion exchange and lower it for cation exchange – but don’t stray too far or the protein won’t like you.
The other way to get better binding is to lower the competition – start your salt gradient at a lower salt concentration (and remember this means you have to lower the salt concentration in your protein sample itself – even if this means ending up with a lot of liquid… In addition to lowering the competition, when you lower the salt you “unshield” the proteins from the sea of charge around them (lower the ionic strength) so it’s easier for opposite charges to find one another – even if those charges aren’t that strong.
Interactions between charged chains & water contribute to keeping proteins dissolved. & like charges repel so when protein molecules are highly charged the same say they can repel each other, so usually a protein is the LEAST soluble at its pI, but this isn’t always the case. Another thing to keep in mind – at high salt concentrations, because the liquid’s so “unattractive” for water-avoiders you get enhanced hydrophobic interactions (basically the charge-avoiders would rather be with other charge avoiders so they stick together.