With general proteases, there’s a time and a place! Normally, I try to keep these “protein scissors” FAAAAAR away from these chromatography columns, which I use to *purify* proteins by flowing them through the columns’ beads. I even proactively destroy proteases before they can destroy my protein by using a cocktail of protease inhibitors designed to target the main types of proteases. But today I (cautiously) added one of the most promiscuous proteases of them all, pepsin, to a column in order to clean it (I’ve done 11 protein purifications in the last 2 weeks, and my column was crying out for cleaning (by giving poorly-resolved peaks – and flowing sooooo slowly)! That generic chewnicity of pepsin which makes it something to avoid *during* purification also makes it great for chewing up any protein gunk that’s stuck on these columns. So today I thought I’d tell you more about types of proteases and protease inhibitors (and finish with some practical advice for column cleaning!).

This cleaning strategy takes a page out of our body’s playbook. The stomach uses hydrochloric acid to help denature (unfold) proteins to make them easier for the protein cutters (including pepsin) – enzymes called proteases. Pepsin does some preliminary protein snipping in the stomach and then, in the intestines, the bulk of the protein cutting takes place, thanks to the enzyme trypsin (which is excreted by the pancreas) – trypsin chops proteins into a few letters long, then more enzymes in & on the surface of intestinal cells finish the job, freeing up individual amino acids. 

In addition to having lots of different proteases, our bodies also have enzymes for breaking down sugars, fats, nucleic acids (DNA/RNA), etc. Amylases and friends help chop carbs, nucleases nudge apart nucleotides, lipase unlatch lipids, etc. This allows us to break down molecules for energy and/or parts, get rid of molecules that have extended their welcome or are damaged, and prevent molecules from entering our cells that were never welcome in the first place (e.g. invading microbes). But today I want to focus on proteases!

jargon note 1: “Enzymes” are usually proteins (sometimes protein/RNA complexes (like the case with ribosomes) or just RNA (we often call such enzymes ribozymes) and they mediate and speed up (catalyze) reactions by doing things holding the molecules together in the right positions for whatever they need to do and providing an optimal environment for the reaction to take place. 

jargon note 2 – “protease” and “peptidase” are often used interchangeably but technically a peptidase specializes in cutting short chains of amino acids, whereas a protease specializes in cutting long chains of amino acids (which in their folded form are better known as proteins). 

We can classify proteases/peptidases in a couple of ways…

  • where they cut: “Endo”protease/peptidase refers to ones that cut in the middle of peptide chains and exoproteases/peptidases chew off the ends. 
  • what cutting mechanism they use (what gives these scissors their blade): 
    • pepsin is an example of an aspartic lprotease – it uses an Aspartate (Asp) amino acid residue to help water attack and break a peptide bond. Glutamic proteases work similarly, but they use Glutamate (Glu) instead of Asp. 
    • metalloproteases use a metal to help the water out
    • In all of those, water is doing the actual work, so there’s no “covalent intermediate” whereby part of the cut protein is stuck to the protease. However, with serine proteases (which I will discuss in the most detail) which use Serine (Ser), there *is* a covelent intermediate.  Similarly for cysteine proteases (which use Cys instead of Ser) and threonine proteases which use Thr instead. So it’s protease + peptide -> proteasepep + tide -> protease + pep + tide (hopefully that’ll make sense later and/or in figs)
    • another terminology note that’s easy to get confused about – when we classify proteases as “aspartic proteases” or serine proteases, we’re talking about what amino acid is in the active site of the protease doing the work. We are *not* referring to where on the protein or peptide the protease likes to cut. (i.e. a serine protease doesn’t prefer to cut next to serine). I wanted to put this here because I’ve heard people think this, which is totally understandable because when we talk about serine phosphates we *are* talking about enzymes that remove phosphates from serine!

Trypsin is one example of a serine protease, but it’s just one of many, all with the same core MO. Basically they all have this conserved catalytic triad of a serine (which directly interacts with the peptide) helped out by a histidine (His, H) & an aspartate (Asp, D), which, through a “charge relay” help get the serine to give up a proton and become more reactive (we’ll get more into this below). Around this conserved core, evolution (as allowed for by gene duplication giving natural selection multiple copies to work with and random mutation providing variation for it to act on) has “played around” with the surrounding parts, allowing different serine proteases to specialize in cleaving at different spots on proteins, and adapting to work in different environments.

This catalytic triad is one of the most beautiful examples of amino acids working together to get a job done! Here’s an overview of what we’re gonna see – when serine proteases cut, half of the thing they cut gets “stuck on them” so you need a second cutter to cut it off. So they use a “ping-pong” mechanism. First, the serine gets activated so it cuts the peptide bond. Now half of the peptide’s stuck to the protease & 1/2 is released. But you can’t use the serine to cut off the stuck part because the serine’s what it’s stuck on! So it brings in a water molecule. And it activates it. Then the activated water frees the serine to do it again.

In order to do all that activating (of serine & water), you need the help of the triad’s other members, His & Asp. You don’t want to activate Serine until go time, so all’s calm until substrate binds to a specificity-determining pocket. This gets the protein to shift a little and this makes Asp’s O- move closer to His, drawing away His’s H, so it’s more willing to steal Ser’s H, giving you an “alkoxide ion” 

Ser really doesn’t want negative charge, so it goes on the nucleophilic attack, attacking the carbonyl carbon of the peptide bond in the substrate. And this might make Ser happy, but it kinda throws everything else out of whack – carbon’s left with a really unstable tetrahedral (bound to 4 things) intermediate – it’s gonna break – but the key is to break it the “right way” – and this is helped out by a “side-pocket” of the enzyme called the oxyanion hole – it helps stabilize this unstable intermediate so that it splits the right way – breaking the peptide bond between the carbon that was the carbonyl carbon and the nitrogen, with the nitrogen snatching off the proton that His stole from Ser on its way out.

Congrats – you’ve just cut the peptide & released part. But, before you start celebrating… you still have the other part stuck on Ser… So now we have to get that off. And this is the “slow” part – because we have to get water in and activated. The His is going to do the activating again, just like it did with Ser (remember that the peptide took the proton from His that His took to Ser, so there’s now room for another proton there!). So the nitrogen of His snatches a proton from water, giving you a hydroxyl ion, which for reasons akin to the reasons the alkoxide ion attacked the carbonyl C, will nucleophilically attack the carbonyl carbon. So you get another tetrahedral intermediate that gets broken by Ser’s O taking a proton from His. 

It sounds like you could make a good kids’ book or song from it…. Ser steals a proton from His that His stole from water to replace the proton that the N-terminus stole from His that His stole from Ser! And with this, the C terminal is released and the enzyme is back to its starting state so it can do it all again. Yes, *now* you may celebrate! (although, who am I kidding – I say celebrate the small things!)

It’s a testament to the efficiency and biochemical beautifulness of this triad that so many enzymes throughout evolution in species far and wide have conserved it (and it’s even evolved separately a few times). But, if all these enzymes cut the same way, how do we bring in specificity? Kinda like how you can’t use a paper shredder to cut an encyclopedia because you stuff an encyclopedia into the opening slot of a paper shredder (and please please don’t try! encyclopedias don’t deserve that!), where a protease cuts has to do with how its specificity pocket is shaped and how welcoming the side chains sticking out into it are to different types of substrates (the thing that has to bind & get cut). 

For example, trypsin has negatively-charged aspartate jutting out into it, attracting positively-charged amino acids, so it cuts the carboxy side next to lysine (Lys, K) & arginine (Arg, R). Chymotrypsin, on the other hand has a big, hydrophobic pocket making it great for cutting next to big bulky amino acids – tryptophan (Trp, W), phenylalanine (Phe, F), tyrosine (Tyr, Y), and leucine (Leu, L). Elastase has a really tiny pocket, so it can only cut chains with small amino acids – alanine (Ala, A), glycine (Gly, G), & serine (Ser, S)

Those guys are pretty generic, which is good for places like the intestines where you’re trying to break down a lot of different proteins (proteases themselves are super hardy in order to withstand the constant threat of scissoring). But if you were to release those generic-ish proteases into a cell, all would go to hell! 

Of course, this raises the challenge of making the proteases inside cells and shipping them to where they need to go to work all without them chopping things up along the way – the solution? – don’t activate them until they arrive at their destination – digestive enzymes are made as inactive precursors called zymogens that have extra amino acids that have to be clipped off before the enzyme is active – clipping which is done by other proteases. For example, trypsin is made as the inactive trypsinogen, which travels safely to the intestines where it gets activated by another protease, enteropeptidase, which cuts off its tail, activating it.  And then trypsin can turn the zymogen chymotrypsinogen into chymotrypsin and proelastase into elastase. A similar strategy is used with some hormones that are made as inactive prohormones and activated by cleavage.

Those cleavages are a lot more specific, which is possible because there are specialized proteases which have a lot pickier active sites. An example of this is thrombin – it’s tasked with cutting a protein called fibrinogen, helping blood to clot. Thrombin recognizes the sequence Leu-Val-Pro-Arg-Gly-Ser & cleaves between Arg & Gly.

These more specific proteases are often used in recombinant protein expression, where we stick a gene for a protein into cells (often bacteria or insect cells) and have them make the protein for us – if we add extra DNA letters onto the gene we can get the cells to add extra protein letters onto the protein, which we can use as a tag to help us purify them. And if we insert a protease cleavage site in between the tag and the protein we can use the matching protease to cut off the tag once we’re done using it so we can study the tagless protein. 

Even the “generic-ish” ones like trypsin & chymotrypsin are useful in the lab. In mass spectrometry (mass spec) they’re used to cut proteins up into short peptides as part of the process of determining their identity. And they’re used in lower amounts for “limited proteolysis” where you add a little bit and track protein digestion products over time to try to figure out which regions are the most flexible, accessible, and thus easiest to cleave. This can be really helpful for identifying protein structural domains (parts of the protein that have distinct, stable, 3D shapes). 

In addition to restricting where proteases cut (both where in the body and where in the peptide), our bodies have another cool way to prevent unwanted peptide cutting – SERPINS – they’re not snakes, they’re “serine scissor safety guards.” Their name comes from SErine PRotease INhibitors and they act as “fake substrates” that get stuck in the protease when it tries to cut them – it’s able to cut them but it can’t release them because, once cut, the serpin can (still bound to trypsin) shape-change, drag trypsin along with it,  and move the trypsin’s active site out of whack in the process.

An example of a serpin you might have heard mentioned in commercials for emphysema treatments is alpha-1-antitrypsin – in the bloodstream, it protects tissues from the serine protease elastase, which a type of immune cells called neutrophils secrete into sites of inflammation to break down connective tissue so that blood cells have an easier time getting in to go to work repairing damage. alpha 1-antitrypsin acts as a sort of “moat” that limits elastase’s area of action so it doesn’t just go chewing up connective tissue throughout your body. But smoking can modify this serpin so that it fails to do its job adequately, so the moat is breached and elastase is able to chew up the lungs. Some people have a genetic alpha-1-antitrypsin defficiency, so they’re more at risk for emphysema even in they don’t smoke. alpha 1-antitrypsin is just one example of more than 30 serpins have been discovered in humans. A couple others are antithrombin, which keeps thrombin in check during clot formation and antiplasmin, which inhibits plasmin so that clots can be dissassembled. 

As you might expect based on there being so many, they’re specialized to work on different proteases so your body can inhibit one without screwing up the activity of others. But in the lab, when we are purifying proteins and want to keep them from getting chewed up along the way, we want to prevent all the chewers, we don’t care about specificity. So we rely on “simpler,” much more generic, types of serine protease inhibitors. 

The biggest place this comes into play is during the cell lysis part of the protein purification -lysis just refers to splitting up and in this case we’re splitting up the cells containing the proteins – (hopefully) *not* the proteins themselves! This exposes the cell insides to whatever’s outside and whatever’s inside the little membrane-bound compartments (some of which contain proteases – this membranous segregation is another way to protect unwanted cutting). Normally the proteins inside cells don’t get in contact with those unless they get an “escort” but when you break open the membranes, you lose that protective barrier. So we want to make sure that as soon as they’re released, they’re inactivated. So we add protease inhibitors to the solution (lysis buffer) that you resuspend the cells in before breaking them open.

We need something that will inactivate the proteases but not hurt our protein in the process. This can be especially tricky because the things we want to destroy are made up of the same stuff as the thing we want to save (amino acids). How can we stop it? We can add something to hide what the protease needs. And a serine protease needs serine. Hide that serine, and it’s like sticking a safety guard on the scissor’s blade. A safety guard we commonly turn to is PMSF (phenylmethylsulfonyl fluoride) which sticks its PMS onto the Ser’s O.

activesite-OH + PMSF -> active site-PMS + HF

PMSF doesn’t “sulfonylate” all the serines, just the ones in the active site, where there’s the perfect environment for it – the same conditions that make it easier to swap serine’s H for a protein part make it easier to swap it for a sulfonyl part. And, unlike removable safety guards, this guy gets glued on with a strong, covalent bond, making it “irreversible.” You can’t just use water to cut it off because the active sit isn’t built for that – it’s exquisitely made so it’ll only cut peptide bonds.

BUT this doesn’t protect our proteins from all proteases – I’ve been rambling on about the serine proteases because I think that triad is so cool (and because I wrote most of that for a post on serine…) but remember how I told you about all those different kinds of proteases, which don’t rely on serine. How do we inhibit those?!

Thankfully, each type of protease has an “Achille’s heels” (though the ones that don’t have covalent intermediates we can only inhibit reversibly since we can’t trick the protease into getting stuck on it).  We often use “protease inhibitor cocktails” which are just a pre-mixed mix of inhibitors.

We make our own but you can buy pre-made cocktails. Several of these protease inhibitors are pretty unstable, so we make it pretty fresh (and keep it frozen – although it doesn’t really freeze at -20 degrees (C) because it’s dissolved in alcohol because some of the protease inhibitors don’t dissolve in water. To get around stability issues, companies sell cocktails in the form of tablets. But if you purify as much protein as we do, that’d be sooooo $$$$$

Note: the most obvious way to inhibit a metalloprotease is by stealing its metal, which can be done quite well by “chelators” like EDTA. So a lot of the commercial cocktails have EDTA in them (but there are also EDTA-free ones). BUT if you’re using IMAC (immobilized metal affinity chromatography), you don’t want EDTA! It’ll de-nickelize your nickel column! And you also don’t want to use EDTA if your protein needs metal.

we don’t put EDTA in ours, but we do put…

  • PMSF – which, as you now know inhibits serine proteases IRREVERSIBLY
  • pepstatin – which inhibits aspartic proteases reversibly 
  • leupeptin – inhibits serine and cysteine proteases reversibly
  • benzamidine – inhibits serine proteases reversibly
  • aprotinin – inhibits serine proteases reversibly

It might seem weird that we have multiple serine protease inhibitors, but some work better for different proteases so we play it safe!

here’s a really great overview: https://www.labome.com/method/Protease-Inhibitors.html 

We only include the protease inhibitors in our lysis buffer – the liquid we break the cells open into. Once we get purifying it, we purify off those proteases and don’t need to worry about adding inhibitors (and would prefer not to have extra stuff around our protein anyway!)

Speaking of purification – and proteases… Back to the story of why I purposefully put pepsin on a column! As I mentioned, I’ve been doing a LOT of protein chromatography, which is where we purify proteins by running them through various columns filled with little beads (resin). Different types of resin have different properties. And different proteins have different properties. So the proteins interact differently and you can separate them. more here: http://bit.ly/meettheakta 

But sometimes, proteins and/or gunk gets stuck on the column and prevents the proteins from flowing through easily. The AKTA system that controls the flow through the column slows it down thermostat-style to prevent pressure buildup. So the more gunked up it is, the slower the flow. So what should be a 40 min or so long run can take a couple hours. This was happening to one of the columns I’ve been using a lot, the MonoS, which is an cation exchange column which separates proteins by charge. So, as I do periodically I gave it a deep clean following GE’s advice (not a paid endorsement obviously because I already forgot that GE sold it to Cytivia or something like that?) Anyways…

I started by running through 70% ethanol (which should help dissolve any hydrophobic (water avoiding) proteins that had crapped out in the column. And then I ran through a lot of water to wash that out. And then I added pepsin – which as we know know is an aspartic protease! I added it in dilute acetic acid to lower the pH to where pepsin’s used to working. I let it flow through until the column was filled up with it and then halted the flow and am letting it work overnight, hopefully chewing up any stuck-in/on proteins. and then I washed those lines and the system really well! Because it’s still kinda scary putting pepsin in there…

cool side story note about pepsin being useful in another way… In 1934, Dorothy Hodgkin’s lab obtained an x-ray diffraction pattern which showed that proteins – even non-fibrous ones – can have orderly structures. And it opened the door wide for protein crystallography.  much more here: http://bit.ly/dorothycrowfoothodgkin

#365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 

Leave a Reply

Your email address will not be published.