On Tannins, Tea, and Soymilk: Episode 2

Welcome back. It's time for the second and final episode of the Thrilling Saga of Tea and Tannins: In Which There Are Pictures of Proteins!

After last week's cliffhanger, we learned that some chemical properties of tannins (pi-stacking, extensive hydrogen bonding) may be responsible for their ability to aggregate proteins (1,2). To provide a visual reference, below I've rendered an image of glycinin, a soy protein (4). In purple are the aromatic residues, or the amino acids that contain planar rings composed of conjugated (alternating) double bonds. These include tyrosine, phenylalanine, and tryptophan, and all are potentially capable or being viciously pi-stacked by the aromatic portions of tannins.

Glycinin: PDB File 1OD5: Rendered in Mac Pymol

I know that thing up there looks like an amoeba with athlete's foot, but I'm trying to demonstrate an idea here, which is that a relevant protein in soy (as well as most proteins, really) has some solvent exposed aromatics. To highlight the residues with the ability to hydrogen bond would be silly, since most do, and even the very backbone of a protein contains hydrogen-bonding amides.

I'm going to throw up another image of a protein, this time a personal favorite of mine. I've rendered it as a "cartoon" in order to show you the very specific, unique structural characteristics it has. Don't worry about details...just take it in.

JMJD2A: PDB file 2Q8C: Rendered in Mac PyMol

This little beauty is JMJD2A, about which I imagine I will post much more in much greater detail in the future. Suffice to say it occurs in the nucleus of your cells, swimming about, tinkering with the integrity of your DNA...your very essence...in ways we scarcely yet comprehend! Anyways. Notice the neatly coiled helices; take in the simple sheets, and the unusual curves. Notice that green thing in the middle. This is where Rule 1 (which I definitely kinda just made up in Episode 1) is beautifully illustrated.

JMJD2A binds very specifically to that green thing because its alarmingly complex structure has evolved the ability to do so. Change one tiny facet of the protein, and its shape and activity may be utterly lost.

For a mathematical approach to why and how proteins fold the way they do, check out this book (5):

Hey! That guy was my professor!

So proteins fold up and stick to themselves, excluding the water from their greasy insides. They also tend to not stick to each other, unless they have been built to do so. In fact, when proteins get out of control and do start sticking to each other, annoying little side-effects happen, like, say, Alzheimer's disease. That's another story.

Perhaps the strongest force that holds proteins in their shape can be visualized with this lovely stock photo of olive oil in water.

Like people, (or me, at least) proteins behave the way they do in order to avoid discomfort. The greasy parts of a protein REALLY don't like touching water, so they fold in on themselves. I REALLY like having a bed to sleep in, so I go to work to pay rent!

..........Then along come tannins.

These buggers are built to screw with protein. The delicate web of interactions that so precariously hold the protein together are interrupted when tannins stick all over their surface. Push the protein too far and BLAM!

When a protein unfolds, its greasy guts are spilled onto the outside. The protein next door isn't in better shape, either. In fact, the whole neighborhood just went up in smoke. These amorphous lumps of oil flounder about, trying their damnedest to escape all the pain. They group. They attack their fellows. Hundreds, thousands, millions of unfolded protein chains stick at random to the guy next to them, unfolding him in the process. An aggregate is born. A herd, if you will.

Robert Kirkman's The Walking Dead. Seriously, read this book.

 Did anyone else go to a zombie place just then? No? Just me?

We could also go vegan, and say tofu is born.

Yes, the woman's soy milk (Remember that? Episode 1?) was denatured by the tannic tea. Like a cooking egg, droplets of oil in water, the brain of an Alzheimer's patient, or a herd of mindless flesh-eating zombies drawn together by the prospect of tasty brains, the soy protein went GLOOOP! and stuck together.

............................

Here is where I mention that loads of things cause proteins to aggregate or denature. Too much salt. Not enough salt. Too acidic. Too basic. Too hot or too cold. Hell, some proteins have evolved to just bide their time for the signal to start aggregating in order to send a message or congeal blood or commit cell-suicide (6).

The way in which tannins specifically accomplish this task is still kind of special. They make your tongue feel dry. They preserve animal hides by linking the proteins together and simultaneously preventing the growth of microorganisms that would otherwise just rot the meat. They warn gorillas which plants are probably not OK to eat. And apparently, they cause my stomach lining to take up arms and revolt.

Unless I add a splash of milk first.

Hope to see you next week, when I plan to go organic on your asses. As in organic chemistry, with mechanisms and everything. If you have a favorite reaction you'd like to see me explore, leave a comment. Reaction must be awesome, or awesomely relevant to something else that is awesome.

Thanks for reading!


1.  Gyemant, G et al. (2008). Evidence for pentagalloyl glucose binding to human salivary α-amylase through aromatic amino acid residues. Biochimica et Biophysica Acta 2, 291-296.

2. Hagerman, AE et al. (1998). Mechanism of protein precipitation for two tannins, pentagalloylglucose and epicatechin16 (4->8) catechin (procyanidin). J Agr Food Chem 46, 2590-2595.

3. Chen, Z et al. (2006). Structural insights into histone demethylation by JMJD2 family members. Cell 125, 691-672.

 4) Adachi, M. et al. (2003). Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer. Proc Nat Acad Sci. 100: 7395.

5) Dill, Ken A., and Bromberg, Sarina. Molecular Driving Forces: Statistical Thermodynamics in Biology, Chemistry, Physics and Nanotechnology. Garland Science, 2010.

6) Shi, Y. (2008). Apoptosome assembly. Methods Enzymology 442, 141-156.

On Tannins, Tea, and Soymilk: Episode 1

I have this problem where I love strong black tea, but I occasionally turn green and vomit it back up. It's...not pleasant. Being stubborn and showing a complete disregard for my stomach lining led me to work through this minor bump in the road. Through trial and error, I found that I only had any nausea from tea if I am on an empty stomach. Not only that, but some foods hanging out in my gut seemed to do the trick, while some provided no protection at all. Milk, it seemed, worked really well. And eggs. Or pretty much any big, balanced meal. Toast and jam: not so much.

So I assumed it was protein, and dissolved protein at that, that buffered my belly from the inevitable puke-party.

Then the other day at work, a woman complained to me of two separate half-gallons of her soy milk spoiling before the expiration date.

"What were you using your soy milk for?" I had my suspicions.
"I put it in my tea."
"And it curdles?"
"It looks like cottage cheese and it sinks to the bottom."

Well, I thought, it actually looks more like tofu, since it basically is. And no, there is nothing wrong with your tasty soy beverage. It's a harmless, but annoying physical process, likely very similar to what was happening in my gut.

Here's my theory: Black tea (and green tea) contains considerable amounts of tannins, a fairly broad class of phenolic compounds so named because they were historically used for tanning leather; these tannins are binding to, aggregating, and generally dicking around with the proteins and mucus layer in my gut. Unless there is another more accessible (dissolved), abundant protein source for them to get to first!

So before I get into what tannins may be doing to proteins in my stomach and in soy milk, let's talk about what tannins are - I mean chemically - and why their structures determine this nauseating (and occasionally useful!) function.

Rule 1: Activity follows structure.

With Rule 1 in mind (which I just decided was a Rule...It seems true in my experience) let's look at a couple of different types of tannins.

Pentagalloylglucose

Pentagalloylglucose (PGG) is the per-esterified gallic acid ester of glucose. That just means at every available -OH group in glucose, a gallic acid group has been appended. It's also a prototypical hydrolyzable tannin, and it is capable of precipitating protein out of a solution (1, 2).

(4->8) Catechin Dimer

The catechin dimer shown above represents a member of another class of tannins, the flavanols. You're probably aware of this class from the ceaseless talk about antioxidants in tea, pomegranate, acai fruit, red wine...blah blah blah. Yes, they scavenge free-radicals, but they're also astringent tannins that are capable of binding to, aggregating, and precipitating protein (2)!

Similar to both classes of tannins? Lots of hydroxyl (-OH) groups, and a number of phenolic hydroxyl groups (an -OH sticking to a benzene).

OK. So. Tannins come in a couple of structural classes. They both have phenolic hydroxyl groups. They can mess with proteins. And they can be found in black tea. To understand how tannins might do this, let's think about the transient, non-covalent bonds a phenol could form with proteins.

In scenario 1, the aromatic ring itself uses its delocalized pi-bonds (benzene's most attractive feature, IMO) to "stack" on top of an aromatic ring on the outside of a protein, like tryptophan, tyrosine, or phenylalanine. In fact, some fancy scientists removed some of the aromatic residues out of a protein that is normally susceptible to tannins and found that the tannin (PGG) lost much of its ability to bind and aggregate that protein (1). Neat trick. What's next.

Scenario 2: Those tannins? They're covered in little polar -OH groups. The short version here is that "O" carries around a small negative electrical charge, and the "H" carries around a small positive electrical charge. There are buckets of residues on a protein which do the same, so you can imagine that enough of these weak, fleeting hydrogen bonds formed between tannins and a protein could become significant.

.....

I should mention how tempted I was to tell the lady with the chunky soy milk everything I've just explained here. Really, I think she would have felt much better knowing about pi-stacking as it relates to her morning cuppa (don't you?). I showed some restraint instead, which is something I scarcely have to worry about here.

In the next segment, I'm going to put the pieces together and get to the point...if there is a point...Oh yeah! How do the chemical properties of the tannins discussed above curdle a tasty vegan milk substitute? Why does tea make me want to vomit? Why are there so many coffee makers in my garage?! Who left the bathroom light on!!!?!


1.  Gyemant, G et al. (2008). Evidence for pentagalloyl glucose binding to human salivary α-amylase through aromatic amino acid residues. Biochimica et Biophysica Acta 2, 291-296.

2. Hagerman, AE et al. (1998). Mechanism of protein precipitation for two tannins, pentagalloylglucose and epicatechin16 (4->8) catechin (procyanidin). J Agr Food Chem 46, 2590-2595.

3. Chen, Z et al. (2006). Structural insights into histone demethylation by JMJD2 family members. Cell 125, 691-672.

Mission Statement

Every molecule has a story. Or at least the good ones do. Sometimes they're full of intrigue and heart-stopping drama, and sometimes the tales they tell just smell kind of nice. Like lavender, or browned butter. I intend this blog to be about those chemicals that have wandered into my own story and enriched it in one way or another.

At my disposal are a handful of old textbooks that survived my many moves around the Bay Area, the internet, a copy of ChemDraw Ultra, Mac PyMol, and my brain. Enjoy.