Using DTT? How could you tell? I recognized that rotten-egg smell! And, while this might normally be a “yuck,” in the days of covid, it’s a nice relief to know that I haven’t lost my sense of smell! Like a mini (and definitely not sensitive/reliable/approved) test. And I do it a lot because I use this REDUCING AGENT a lot to keep the REDOX environment in my artificial buffer sea similar to that intracellular proteins see. And I want to tell you about it – and others. But, can the bumbling biochemist reduce REDUCING AGENTS into a post? Maybe if she focuses on the ones biochemists use most – DTT, TCEP, & BME. To understand why we use them, let’s go for a dig into the redox world of OIL RIG!

OIL RIG is a mnemonic we use to remember Oxidation Is Loss (of electrons) and Reduction Is Gain (of electrons). Electrons are negatively charged but positively cool! They’re little subatomic particles that whizz around the dense, positively-charged, nucleus of atoms. 

Different atoms have different numbers of electrons. They keep most of them to themselves, held in by the positively-charged protons in the nucleus, but the electrons furthest away from the nucleus (valence electrons) feel the pull less, so they can play the field more to get to their “ideal”

Atoms have “ideal” numbers of valence electrons – for a lot of the molecules we talk about in biochemicals (like carbon (C), nitrogen (N), oxygen (O) & sulfur (S)) this number is 8 – leading to the the so-called “octet rule.” Hydrogen (H) is too small for that many electrons – it only has a single proton, so there’s no way it could keep a hold on 8! Instead, it shoots for 2. If you look to the right-most column of the periodic table, you see the ones that already have their ideal number, the “noble gases.” All the elements want to be like those.

For some atoms, losing electrons gets them closer and for others, gaining electrons gets them closer. So, in “rig-ged reactions” the molecules conspire so each gets what they want. 

Atoms can lose electrons – which we call oxidation – the OIL in OIL RIG. And when they do, the electrons have to go somewhere – and the somewhere they go to is another atom. So that other atom gains electrons, and we call that reduction – the RIG.

Together, we call these redox reactions and you can’t have 1 without the other. 

One molecule, the reducing agent, or REDUCTANT “wants” to get rid of electron(s) and the other molecule, the oxidizing agent, or OXIDANT “wants” to take them.⠀

But once the reductant gives the oxidant the electron, the reducer becomes oxidized and once the oxidant takes an electron it becomes reduced and reluctant to act as an oxidant!⠀

So it’s not like something’s always in the “I want to reduce” state or alway in the “I want to oxidize” state. Their “mood” depends on their “redox environment” – the amounts of reductants and oxidants around them. 

Our cells maintain a reducing environment in part to protect against oxidative damage. Oxidants like reactive oxygen species (ROS) can break up existing molecular relationships, doing things like breaking DNA if they’re not kept in check. 

Cells control the redox environment by stocking up on reducing agents like glutathione (you might remember this from my post on GST-tagged proteins). There, we use glutathione to compete for binding to a GST tag, not for its reducing properties. 

Glutathione (made from 3 protein letters (amino acids) – Glu, Cys, & Gly) acts as a “Redox buffer” – it goes back and forth between reduced (GSH) & oxidized forms(GSSG) where there are 2 glutathione linked by disulfide bond.

That “disulfide bond” (aka cystine crosslink) is between the “thiol” side chains of the amino acid cysteine. That was a lot of jargon, sorry – here’s what I mean…

Proteins (molecular machines) are made up up of chains of building block “letters” called amino acids that are like charm bracelets. Amino acids have a generic backbone (chain link) that allows any amino acid to connect to any other amino acid as well as a unique side chain “charm” that sticks out. Charms have different chemical properties that allow them to interact in different ways w/one another (important for the protein to fold properly) & w/other molecules (important for intracellular interactions).

The reducing power of glutathione comes from the Cys, whose “charm” is a -CH₂-SH group. Cys’ side chain is a -CH₂-SH group. -SH is called a THIOL (it’s alcohol’s (-OH) sulfur CYSter).  More on alcohols here: http://bit.ly/2QWKWXp but it just refers to molecules having hydroxyl (-OH) group(s). So not all alcohols are “alcoholic” in the social outing sense. Anything with an -OH is an “alcohol” in the chemistry sense, so things like sugar are super alcoholic. 

Cysteine isn’t gonna get you drunk, but the thiol can lead to some protein personality changes! 2 Cys (either within the same protein or between 2 proteins) can link together (protein)-SH + HS-(protein) 👉 (protein)-S-S-(protein) to give you a  DISULFIDE BOND or “cross-link” between 2 Cys residues in the same or different proteins

-SH + -SH ⇌ -S-S-

Unlike other charm-charm interactions, which are just *attractions* based on charge (or partial charge) differences, this is a covalent bond, so it’s strong (and good for sturdying-up secreted proteins that have to live outside the comfort of the cell). For example, pairs of disulfide bonds are used to keep the 2 separate peptide chains of the hormone insulin connected as it travels throughout your bloodstream to tell cells to let in and use glucose (blood sugar). 

BUT these Cys-Cys bonds aren’t quite as strong as the covalent bonds in the protein’s backbone (linking the chain links) so you can split them back up without splitting up the chain links. This splitting & unsplitting involves REDuction & OXidation (REDOX) reactions. More here (http://bit.ly/2yBOeDi) but basically they involve thing 1 (the oxidant) giving e⁻ to another thing 2 (the reductant), reducing thing 2 & oxidizing thing 1 in the process

remember OIL RIG: Oxidation Is Loss (of e⁻), Reduction Is Gain (of e⁻)

When cysteine’s link up as cystines, they share their “extra” e⁻ so they’re “losing” e⁻ (being OXIDIZED) (lose the “e” in the name (and also “e⁻”)). And when they split up, they get the e⁻ they’d been sharing “back”, so they’re REDUCED

Which state Cys is in depends on the “redox environment” (whether there are more reductants or oxidants around) and keeping the right redox conditions is really important for protein shape & function because, since these bonds are stronger than the normal intermolecular bonds, they’re harder to break up, so it’s important you get it right the first time. Because you might not find the right partner if you have to do it again…

If a cysteine residue gets reduced, it gains electrons – and the negative charge they bring – so it becomes nucleophilic – seeks out the positive charge coming from a neighboring atom’s nucleus. So you want to make sure the neighbor it finds is the neighbor it’s supposed to bind! Inside your cell, conditions are kept “reducing” thanks to glutathione “buffering” so proteins that do have vulnerable disulfide bonds that need to be kept go through alternative processing pathways where they’re made & shuttled out of the cell (into the oxidizing extracellular environment) in protective membrane-bound vesicles.

An oxidizing state in your cells can thus be “dangerous” because inappropriate bridging can occur if they get oxidized by reactive oxygen species (ROS). Low, CONTROLLED, levels of ROS are important for things like signaling but high levels can be damaging because, as their name suggests, they’re reactive and they can react with things like DNA & proteins, messing them up. So your cells control the redox environment by stocking up on reducing agents like glutathione. Glutathione is made from 3 protein letters (amino acids) – Glu, Cys, & Gly & acts as a “Redox buffer.” It goes goes back and forth between reduced (GSH) & oxidized forms(GSSG) forms, where 2 glutathione are linked by a disulfide bond.

When we purify proteins, we usually try to keep them in an environment that’s close to what they’re naturally found in. Evolution’s had years and years of natural selection to adapt these proteins to be happiest and most active in that environment. If you’re dealing with an intracellular protein that’s used to that reducing environment and you change things up to an oxidizing environment, proteins can panic like it’s the end of the world & hook up with the nearest available thing instead of waiting for their “soulmate.” 

When we’re studying proteins that are normally inside of the general “inside” of cells, the environment we’re trying to mimic is that of the cytoplasm. So we want to keep a “reducing environment” – so we also usually add a reducing agent, which can sop up extra electrons. Instead of using glutathione, which oxidizes too easily, a few common reducing agents we use in the lab are DTT (DiThioThreiotol), TCEP ((Tris(2-Carboxyethyl) phosphine hydrochloride)), & BME (β-mercaptoethanol, aka 2-mercaptoenthanol) (more on them in a second). 

There’s a lot of other stuff going on in the cytoplasm too, and we don’t want to add too much complexity to our buffers, especially since the actual contents of the cytoplasm varies from cell type to cell type and even cell to cell. 

So, in our mimicking we try to stick to the minimal amount of things that will make our protein happy, soluble, and active. 

The artificial “seas” we put our proteins in in the lab are called BUFFERS. The name refers to the pH-stabilizing component we put in there. 

pH is a measure of how acidic (proton-rich) or basic (proton-poor) a liquid is. Neutral’s 7 – lower’s more acidic & higher’s more basic. Buffers are molecules that can give and take protons to keep pH constant. Some ones we often use are Tris, HEPES, and sodium phosphate, and you can learn more about them here: 

We usually buffer the pH around 7.4-8, which reflects cytoplasmic conditions.

Another thing we need is salt. A couple common salts we use are NaCl (sodium chloride, aka table salt), KCl (potassium chloride)

And now, as promised, the reducing agent: A few common reducing agents we use in the lab are DTT (DiThioThreiotol), TCEP ((Tris(2-Carboxyethyl) phosphine hydrochloride)), & BME (β-mercaptoethanol, aka 2-mercaptoenthanol). They all can reduce disulfide bonds, but they also have some different properties that make them better or worse in different situations. 

SMELL: DTT & BME both have that rotten-eggy smell because they both have sulfur in them (the “thio” and “mercapto” give this away). And if you don’t like the smell of DTT (which I don’t think is that bad though maybe I’m just use to it) you don’t want to smell BME – it’s even worse! But TCEPT doesn’t have sulfur so your nose is safe.

COMPATIBILITY: 

  • EDTA turns Ni-NTA resin white and NiSO4 turns it blue – but if your IMAC resin turns brown, you likely have DTT hanging around. 
    • DTT can turn Ni-coated resin, like the Ni-NTA we use for IMAC, brown – this is because it can reduce Ni. Unlike EDTA which actually removes the Ni from the column, this “browning” usually doesn’t actually interfere with protein binding because it’s interacting with the Ni that’s “uncoordinated” – only loosely bound. But it can – and it’s not pretty – but is pretty disconcerting… A way you can use DTT (at low levels) with this resin without it turning brown is to, before you let the column see DTT, pre-wash it with a buffer with a high concentration of imidazole. The imidazole will wash that loose Ni from the resin. (and you don’t really want that loose Ni there anyway right?)
  • TCEP absorbs less light at 280nm (one of the wavelengths we use to measure protein concentration)

size: size-wise TCEP > DTT > BME. You can tell by looking at their molecular weights (M.W.) which tells you how much 1 mole (6.02e23 molecules weighs (in g). TCEP has a M.W. of 250.15 (250.15 in 1 mole), DTT’s 154.25, & BME’s 78.13. Why care? Because TCEPs bigger it has a harder time getting to disulfide bonds that are present “inside” a protein – so it can readily break up intERmolecular bonds (between different proteins) and thus break up dimers but it leaves intRAmolecular bonds (within a single protein) so it doesn’t make them unfold. But, as long as you don’t crank the amount of reducing agent too high (as long as you’re keeping reducing conditions to those like the ones in the cell) you should be ok on this front regardless – if your protein’s that sensitive it wouldn’t be able to stay folded in the cell. 

Speaking of which, proteins that do have vulnerable disulfide bonds that need to be kept go through alternative processing pathways where they’re made & shuttled out of the cell (into the oxidizing extracellular environment) in protective membrane-bound vesicles. 

COST: TCEP is much more expensive. It might not seem like a big deal price-wise if you don’t need much, but when you have to do things like dialysis where you need liters and liters (I had to dialyze against 8L the other day) that price difference really adds up

STABILITY: DTT’s not very stable, so we usually add it “last minute.” What I normally do is, for buffers I make a lot, I make a 10X stock. Then when I want to use it, I just dilute it 1:!0 to get to the working concentration (1X) and add DTT then. TCEP isn’t very stable in phosphate buffers – esp. at neutral pH. 

random other tidbits on these reductants:

  • DTT’s also known as Cleland’s Reagent – which I didn’t know about until I saw it on the bottle one time
  • TCEP’s charged so it can lower your pH a bit. 

We want to avoid new bridging in protein preps, but breaking and reforming disulfide bonds is actually how perms work which segues us into a hairy tail.

Hair – yup there’s lots of cysteine – and cystine – there!. Inside each strand of hair there are actually lots of strands and each of those strands of the protein KERATIN. And it has lots of cysteines. The protein KERATIN gives your hair & nails strength  & it get’s *its* strength from disulfide crosslinks holding strands of keratin together, Each strand has lots of Cys, so lots of ways to link & the “choice” of way determines the curviness of our hair. Keratin’s stretchy, so you can physically force your hair to curl, but it will de-bounce back unless you use some “chemical force.” in a perm, you physically force hair into the shape you want (e.g. curl it around curlers), then apply a reducing agent to reduce the crosslinks along with heat to remove the non-covalent bonds ⏩ the strands can now stabilize in new positions ⏩ add an oxidizing agent to make new cross-links in the new position. A similar function is carried out in our body by an enzyme called protein disulfide iosmerase (PDI) which catalyzes the shuffling around of cross-links to correct improper bonds that would stabilize a misfolded form

If you want to break bridges, you can add a reducing agent. And if you want to build bridges, you add an oxidizing agent. Ones commonly used for hair perms are ammonium thioglycolate as a reductant to break the bonds & hydrogen peroxide as the oxidant to reform them – when you’re ready! (e.g. after you’ve wrapped hair around curlers)

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 

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