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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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In the Pipeline

« Vox Populi | Main | More on Voodoo »

June 15, 2006

And 0.04 Molar in Eye of Newt. . .

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Posted by Derek

You know, I mean no offense to all my pharmacologist friends and readers, but. . .do y'all really know why all those things are in your buffers and solutions? I've been wrestling with this the last few days, trying to straighten out my "vial thirty-three" problem, and it's been interesting.

There's some reducing agent in there, naturally. Can't have those thiols turning into disulfides and balling up the protein, I understand - but does something bad happen if it's not in there? Generally, no one finds out, because, hey, why mess with it? And there's some EDTA, and some salt, and their function is? Well, as far as I can tell, they're also in there because they've sort of always been. Same goes for the squirt of detergent (Brij-35 or some such), and the tiny bit of bovine serum albumin, of all things. It's just part of the old-fashioned recipe from Grandma's Protein Kitchen.

Now, organic chemistry has a little of this, true, but it hasn't reached quite the Ancient Runestone levels of enzymology. We like to use tetrahydrofuran (THF) for a lot of organometallic reactions, for example, but at least we know that that's because THF is a good co-ordinator to metal cations. At least we don't have six other trace constituents in there that we always use whether we need 'em or not. Another example is how we tend to stick to good ol' ethyl acetate and hexane to run TLC plates, rather than look into other solvent combinations that might do a better job - probably because there are just too many of them to investigate, and EtOAc/hexane works well enough.

And that, I think, is the problem that the biologists face. Biochemical systems are tricky. They have way too many variables, which means that their degrees of freedom have to be reduced just to get anything to work. So all sorts of recipes and rules of thumb are handed down. Not all of them are optimal, but they're mostly decent and will allow you to get on with the project without wasting too much time. Especially in the early part of a project, an immediate 70% effectiveness is worth a lot more than a 98% that would take you a month of work to tweak up to.

Comments (27) + TrackBacks (0) | Category: Drug Assays


COMMENTS

1. Massspecgeek on June 15, 2006 9:43 PM writes...

Being a chemist, I can't account for their potion ingredients either. However, I can tell you that if you are analyzing your vial thirty-three samples on an instrument with a little hummingbird on it, you definitely don't want any detergent in it -- especially the oligomeric NPs like Brij, Triton, etc. They have a tendency to make a mess of the spectra and they are difficult to get rid of by RP-LC.

You probably already know that reduced proteins fly better in electrospray because they are unfolded and have more sites available to be protonated. I'm sure that isn't the reason for the DTT/TCEP/etc in the enzymology recipes though.

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2. qetzal on June 15, 2006 9:55 PM writes...

Reducing agents prevent aggregation due to intermolecular disufide bonds. EDTA, at least in some cases, is to chelate divalent metal cations. Sometimes you want that with a protein, sometimes you don't. EDTA has modest antibacterial effect as well.

Some salt is generally essential b/c most proteins will denature if the ionic strength is too low.

Detergents help prevent aggregation by reducing hydrophobic interactions between protein molecules, and also help prevent adsorption of protein to plastics (which can be a major problem if the protein concentration is dilute).

BSA is a bit more voodoo-ish, but probably functions similarly to detergents. The empirical observation is that many proteins are unstable and denature if you dilute them too much. They only seem to be happy if there's a certain amount of total protein around. So if you can only prepare a dilute solution of your protein, adding some BSA to increase total concentration can be a good way to keep it from unfolding.

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3. Pharmachick on June 15, 2006 10:16 PM writes...

I'm with qetzal on the explanations, particularly the part about over-dilution of proteins. The little fellas like to hang out with their friends.

Also, while you chemical fellas (and fella-esses) are generally more interested in structure, when it comes to proteins we're often more interested in function... sorry. By the way, most of us know that our voodoo buffers make your ability to play with structure difficult, it doesnt stop us from simultaneously making demands like "and while you're at it I think if you could just add a tri-phenyl phosphine to [insert a hideously hindered group] we'd be real happy as we'd like to get it into the mitochondria"

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4. NJBiologist on June 15, 2006 10:48 PM writes...

Here are some guesses for you... BSA, in addition to doing the things Qetzal mentioned, can keep small molecules off glass and plastic. And some enzymes really love their salts. To the point of not working optimally, or at all, without their salts. Particular salts. With a distinctly non-linear concentration dependence.

Now, I'd like to ask some questions... you're sure that EDTA isn't chelating anything important to your reaction, right? And the BSA you've been using--it's the same prep for each of your sequence of experiments, right? When one catalog lists 51 variations of BSA (differing in purity, and preparation, and who knows what else), it's hard not to think about all the other things in cow blood which could co-purify and maybe make a mess of an experiment.

Good luck with vial 33.

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5. GC on June 15, 2006 11:37 PM writes...

I agree with NJchemist on the BSA. The explanation I was always given was that certain proteins (and nucleic acids, although we rarely use BSA with those) are notorious for sticking to the Si-0H groups. So it's either plastic (poor UV cutoff, but the polymethylmethacrylate cuvettes are pretty good), silanized glass (a pain and really not all it's cracked up to be, I try to only use it when I'm deprotecting homegrown oligos, which means they have odd functional groups anyway), or BSA. As for the EDTA/DTT/BME, yeah, same thing. For nucleic acids we almost universally run our gels with 8M urea. This doesn't even stop everything from folding. Certain G-quadruplexes will stay folded even in 8M urea on a cool gel (PAGE, the small 15W ones will sometimes keep them folded, the big 55W sequencing ones often will unfold them, I assume this is just KbT energy doing this since I have no other explanation). This is even for quadruplexes with a relatively low (sub-50C) Tm. BME/DTT (usually bme) helps with proteins and thiolate nucleoside analogues.. It's a mess. Trust me when I say at least biophysical people fret about this to no end. It only becomes a real pain for NMR, since most of this stuff absorbs only at very blue photon energies.

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6. Morten on June 16, 2006 12:45 AM writes...

Like massspecgeek says, DTT will reduce disulphide bridges which in some cases lead to unfolding. I guess in mass spec you really want to reduce the number of covalent bonds as much as possiple and get a nice liniar polypeptide but in enzymology (cute word for anything dealing with proteins) you generally want to keep things somewhat like their physiological state. So generally DTT or something similar when working with intra-cellular proteins and no DTT when using extra-cellular proteins. Trickier when working with membrane proteins - I don't actually know what my collegues do.
If your protein isn't a membrane protein you would probably be better off without the detergent. Which means a new purification 'cos it is pretty much impossible to remove completely.
But when it comes to detergents there are millions of choices so just tell the bio people what you want and what you don't want...

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7. secret milkshake on June 16, 2006 1:31 AM writes...

for detergent you cant try some Tween - these are UV-transparent, non-ionogenic and edible (our PK folks use them in intravenous formulations in whopping amounts - like 10% Tween + 10% DMSO + 80% of water as an injectable solvent base).

Tweens dissolve in water very sluggishly - it really helps to dilute them first with equal volume of DMSO.

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8. Derek Lowe on June 16, 2006 7:01 AM writes...

Thanks to everyone for all the suggestions. For me, the BSA was the most puzzling ingredient in some of these things - everything else I could think of a reason, but not that one.

Pharmachick's comment about optimizing for function is one of the fundamental reasons that chemists and biologists sometimes have trouble talking to each other. We're used to knowing exactly what our compounds are, and even then we often can't explain why they act the way they do. So the relative fuzziness of biochemical systems really scares us - but to someone optimizing an assay, hey, if it works, it works, and never mind exactly why.

Secret, Tween (in my experience) is fine until you get to dogs, some of whom have very nasty reactions to it. I don't think our PK people would let you get close to one with a syringe that had 10% Tween in it.

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9. Novice Chemist on June 16, 2006 7:13 AM writes...

kBt energy? What's that?

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10. Kay on June 16, 2006 7:35 AM writes...

Dear Derek, since chemists like to understand what they do, care to discuss how med chem practices such as assigning drug-likeness have a lower voo-doo quotient?

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11. Kip on June 16, 2006 8:24 AM writes...

Novice Chemist:

k_b T = boltzmann's constant times temperature, i.e. "stat mech" shorthand for the thermal energy of motion. 10kT-100kT is in the kCal range...

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12. Kip on June 16, 2006 8:29 AM writes...

Oops, I need to add that k_b T is implicitly "per molecule", so 10-100kT per molecule is in the kcal range. roughly.

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13. MolecularGeek on June 16, 2006 8:55 AM writes...

Re: 10

Kay,
I'm not Derek, and this may be throwing rubidium down a drain, but I think I see where the difference lies.

Calculating drug-like (or lead-like for that matter) indices is not voodoo, just highly dependent on empirical parameters that are used to generate a number that has a value whose meaning is open to a LOT of debate. Now, I will concede that the generation of those parameters is a dark art unto itself, and that process is highly influenced by one's choice of theoretical model. When things get down to that level, one starts to leave chemistry in the background and migrate to a field akin to medevial theology. But I digress. Any parameterization process involves a certain leap of faith and a little good fortune. I've certainly had to wave a rubber chicken over my spreadsheet from time to time to get things to work out right. (Oddly enough, the chicken also does wonders for clearing up system software conflicts).

Having said all that, I think that most chemists would say that drug-like indicies have a lower "voodoo" quotient because they are based in molecular structure (topological or geometric). With all due respect to biologists (including my fiancee who may be reading this), I can say that reading pharmacology texts and literature always makes me, as a chemist, feel rather uneasy. You get stylized flowcharts and graphs with nice error bars, and you see acronyms thrown around, and then you see that the acronyms stand for things like "methionine decarboxylase activating substance 2" and no mention of molecular structure or composition is made, Maybe, if you are fortunate, a note will be made that this is a 1.3kd polypeptide that appears to be cyclic, or some such. It's certainly possible to do good science on this level, and I wouldn't want to do away with pharmacology or toxicolgy or any other related field because they don't delve into thse issues, but to a chemist, especially an organic chemist, who has atomic level issues drilled into their head from essentially their first day of their first class, it certainly can be a little disconcerting, if not frightening, At the least, it is frustrating because the discussion stops short of where the "real" action is,

MG

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14. GATC on June 16, 2006 9:40 AM writes...

I have found today's postings incredibly amusing! I guess it all depends on your age and training. I consider myself very lucky in that I was brought-up as a microbial geneticist in the early 1980s back before the "kit revolution". That was when all we had was the first edition of Maniatis, a few buffer stocks, and one actually had to know about things like pH, ionic strength, reducing conditions and the like. Now all one has to do is go down to the stock room, buy a kit, and follow the recipe. We are fortunate in that the majority of the folks who INVENTED the "recombinant-DNA revolution" were old school microbiologists and bacterial physiologists who understood basic biochemistry and bacterial physiology. The cytoplasm of a bacterial cell is one of the strongest reducing environments known to science. Restrictions enzymes and other critical reagent enzymes come from bacteria, so naturally beta-ME or DTT is present in storage solutions and reaction buffers. BSA is a general stabilizer and wetting agent; rather important when working with purified enzymes and pipetting in the one (or less) microliter range. It's also better to have contaminating proteases chew on that BSA (usually present in great excess) than your enzyme. Enzyme stocks have 50% glycerol so they don't freeze solid at -20C (multiple freeze-thaw cycles can be a bummer for activity). Purified DNA is subject to spontaneous metal cation-induced hydrolysis so naturally storage buffers have a bit of EDTA (~0.1mM)in there for stability (not too much though because many enzymes involved in DNA manipulations require Mg2+ too!). So you see young Jedi knights, usually there are valid scientific reasons for those mysterious components in your storage and reaction buffers, you just have to THINK and READ, or better yet, take a good seminar-level bacterial physiology course (assuming you can still find one). This would help separate the "bucket-biochemists" from the "roto-vac jocks".

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15. Jim Hu on June 16, 2006 10:48 AM writes...

Derek,

Most of the stuff you're talking about is there to keep your protein from falling out of solution in the in vitro assay of a purified enzyme. I assume that you omit that by the time you move testing to tissue culture cells, much less mice or dogs. and If you need Tween or BSA to solubilize the drug candidate, then the voodoo is back on the med chem side.

Proteins in cells (and intercellular fluids) don't need Tween, or BSA, or EDTA. It's when we take them out of the complex environment they evolved in that they need these helpers. In many cases the assay you're working in is, when compared to the cell
- Too low in total protein.
- Too high in the protein you're testing.
- Lacking chaperones and energy sources to deal with refolding
- Lacking partner proteins that hold your target in a complex
- In nonphysiological salt (example, chloride is not a major anion in cytoplasm)
- Missing the piece of the protein that anchors it to some membrane
- etc.
The chemists are correct that we biochemists/biologists don't know everything about the chemical microenvironments (plural) where proteins evolved to work. But even as we improve our knowledge, you aren't going to do your assays in bioreactors that have concentrated reconstituted cytoplasm. The whole point of assaying purified enzymes is to get away from that kind of complexity. If you want the complexity, you use cells and animals.

There are both scientific and historical reasons for BSA as the nonspecific protein that's sacrificed to surface adsorbtion and contaminating proteases, btw. As something that's a major serum protein, it's soluble at relatively high concentrations. It's also relatively cheap to make and prepare in various levels of usable purity. As #4 pointed out, there are lots of flavors of BSA you can buy. The differences are in how the preps were done. More purity, higher price.

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16. mickj on June 16, 2006 11:33 AM writes...

Serum albumin is the body's garbage collector. It is an efficient "sponge" for a lot of drugs - especially those that are hydrophobic and negatively charged (anyone seen what happens to ANS in the presence of BSA?). I avoid BSA like the plague when investigating ligand-receptor interactions. I recommend the use of bovine gamma globulin (BGG) instead of BSA wherever possible. It is cheap and avoids all the nasty surprises that BSA (in its many states of purity) has up its sleeve!

Mick

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17. orgo on June 16, 2006 11:44 AM writes...

Organic chemists have a few voodoo stories of their own….I have been told that it used to be well-known that THF purchased from Company A produced 1,4-products when gringards were added to a,b-unsaturated enones, while THF from Company B produced the expected 1,2 products. The difference….Company A distilled their THF in copper tubes. For the non-synthesis types, copper catalyzes 1,4 additions. Apparently, there were trace impurities in the THF that catalyzed the reaction.

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18. Chrispy on June 16, 2006 2:34 PM writes...

I agree with most of what has been said here, but I have to add that typically chemists are dealing with molecules which are a good deal higher purity than the ones biologists handle. Even if you express and "purify" a protein it often comes along with cellular baggage, most aggravatingly a low level of proteases which will chew your protein up over time. As GATC alluded, this is one of the roles of BSA (in addition to keeping your protein from sticking to the vial). I suspect that this is also why EDTA was included, to knock out the metalloproteases. I don't like it as a component in a "general" biological buffer, though -- something like 20% of enzymes are metalloenzymes and you'll knock them out, too...

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19. Pharmachick on June 16, 2006 6:35 PM writes...

Round of applause for GATC please. I am somewhat younger than the poster but was "bought up" in a system/lab with very little money. I actually ran my whole PhD project INCLUDING consumables on $7000 + scroungeables for a year... and that included buying a $900 column.

What it meant was we couldn't afford kits so we had to make and optimize everything ourselves... which meant I learned all sorts of interesting things about the way that the assays work (or dont) and what the salts, BSAs, pHs etc of this world are really for. We used to go around chanting "Maniatis is your friend" (alternated with "better living through Pharmacology").

An example: in the days before alamethacin, Brij or Tween were commonly used to activate a certain class of Phase II enzyme. We didnt have any. Yours truly subsequently found you could use up to 2% BSA and get the same activation (I dont really know why but I think everyone here agrees BSA has some detergent-like capabilities). However, above 2% and you got a heck of a drop off. I also found over a period of a few weeks of unrepeatable experiments that you can't store a buffer with BSA in it in glass bottles (for all the reasons listed by posters above).

I have recently had a grad student say (and I quote) "We're out of monobasic Sodium Phosphate but I didnt think it was worth ordering any more, we'll just order the kit next time." SIGH

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20. Novice Chemist on June 16, 2006 6:43 PM writes...

A nice part of academic organic chemistry is that there aren't very many kits and there isn't much money to buy kits. Making reagents using established procedures allows a graduate student to learn technique and save money.

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21. Abel Pharmboy on June 16, 2006 8:35 PM writes...

Like GATC and the other middle-aged folks, I came onto the scene just a hair before kits. I enjoyed asking grad students on their quals about how their kits worked - it illustrated how essential it is to know the composition of the reagents and the reasons why.

Made me recall a late 70s/early 80s story from my old chairman whose student switched reagents and could swear that his divalent cations were being chelated. The supplier insisted repeatedly that there were no chelating agents in their enzyme prep. When the lab finally got a cert of analysis, there was "Versene" at a high concentration. Versene is an archaic name for EDTA.

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22. Courtney Hodges on June 17, 2006 12:34 PM writes...

Am I being overly sensitive, or do biologists and biochemists take a lot of flak for "not understanding" our own techniques?

I wouldn't have the cojones to question the particular reagents used in chemists' experiments/syntheses; I assume they ought to know their field's materials and techniques better than I do. Unfortunately, not many of us biochemists get to enjoy the reciprocity that I think we deserve in that regard.

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23. Novice Chemist on June 17, 2006 2:15 PM writes...

Courtney:

It's just the 'up and down' of science, in terms of where the field came from. Chemists, doubtless, get flak from physicists for not really understanding the nature of the molecules they work with. Physicists likely get torched by mathematics for doing math poorly, etc., etc.

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24. Derek Lowe on June 17, 2006 10:15 PM writes...

As in Ernest Rutherford's line about how all science is either physics or stamp collecting. . .

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25. daen on June 18, 2006 1:24 PM writes...

There are other bits of seeming voodoo in molbio. For example, I kind of understand why it's done now, but using herring sperm DNA for anything other than making more herring seems somewhat ... perverse.

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26. Milo on June 18, 2006 2:45 PM writes...

re #17,

Orgo, just read Derek's post of chromatography solvents. Talk about voodoo! I sometimes do a little dance while pouring my silica slurry hoping that that makes the separation somewhere close to the TLC plate. Oh yes, organic chemistry is filles with voodoo....

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27. Anonymous on July 12, 2007 8:38 PM writes...

Cool...

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