How do you fold a protein, why are chemists obsessed with subatomic particles and how is this related to tofu?
These and many other questions – like why you shouldn’t make kimchi in a metal container or how you can get rid of a corpse – will be answered in this post about vegan food chemistry and tofu. But first a quick disclaimer. Chemistry, biology, and biophysics – the topics of this post – are very complex. In writing this post, I made a lot of simplifications. While the things I write here are true, there are also many other factors playing important roles in these processes that I have omitted for brevity.
What is pH? Like really?
Let’s start with pH. This ought to be familiar from some half-forgotten chemistry course you took a long time ago. You probably recall that pH has to do with acids and bases. A liquid with a pH value of 7 is said to be neutral. A pH below 7 is acidic and pH above 7 is basic or alkaline and so on. Simple. Examples of acids are sour things like vinegar and lemon. Basic or alkaline liquids include things like detergent, lye (drain cleaner), and baking soda dissolved in water [1].
But what does pH actually measure? And what does 7 mean?
The pH scale was first invented to aid the noble craft of beer making. No, really. It was invented in 1909 by Søren Sørensen at Carlsberg brewery in Denmark to help measure the acidity of their beer to aid production [2].
The pH scale is a shorthand for stating how many protons there are in a liter of a sample. A pH value of 7 means that there are 60,221,407,600,000,000 (60 quadrillion, or 6∙1016) protons floating around in one liter of liquid. (See sidebar for this strange calculation.)
This sounds like an awful lot of protons. But one thing I’ve learned from my wife and her work is that when looking at these mind-boggling numbers, it is good to put them in perspective. In the same liter of water, there are 55 moles of water, or 33 septillion (3.3∙1025) molecules. Or in other words, for every single proton, there is one billion water molecules. That is the same ratio as 10 people compared to the total population of the Earth. Now 60 quadrillion sounds awfully small instead.
NB: I write protons here but in reality, these protons quickly pair up with water molecules (H2O), creating hydronium ions (H3O+). But when there is an opportunity for reaction, the protons are quick to take it.
To describe the number of molecules in a sample, chemists like to use moles. This is nothing more complex than a dozen. If you buy a dozen vegan donuts, you will get 12. If you have one mole of molecules in a sample, you have 6.022∙1023 (~600 sextillion) molecules. Simple.
Chemists love to talk about how many moles they have – after all, talking about how many sextillion (1021) or septillion (1024) molecules you have quickly becomes tedious and confusing.
The pH scale just adds an extra step to this. 60 quadrillion molecules is 0.0000001 moles, or 10-7 moles. Instead of saying 10-7 moles, chemists just take the exponent, reverse the sign and say pH 7.
Or as an equation:
pH = – log([H+])
But why protons? And how do they dissolve metals?
That answers the question of what pH is. But why are we so interested in protons? And how does an excess of protons make something corrosive enough to get rid of corpses and dissolve metal?
The answer is that the lone protons are very good at stealing electrons from atoms and molecules or latching on to places where there are free electrons. For instance, when you submerge a metal in a strong acid, the protons will steal single electrons from the metal, and become hydrogen atoms (one proton and one electron, most simple atom there is). These hydrogen atoms will quickly pair up to create hydrogen gas (H2) and bubble away as a flammable gas. The metal atoms that lost their electrons become positively charged ions that are water-soluble and the metal dissolves. This is why many recipes tell you not to use a metal fermentation vessel: even weak acids will corrode metal this way to some extent. This same electron theft is also responsible for the destruction of biological matter, such as corpses. Chemistry World has a good write-up of how corpse disposal works with acids and bases [3].
What about high pH?
While a low pH value means that there is an excess of proton-water molecule pairs (H3O+), a high pH value means that these are scarce. Instead, we have a surplus of hydroxyl ions, OH– (water molecules that have lost one proton but kept the electron). rather than stealing electrons, hydroxyl ions steal protons and become water. Or, they latch onto positively charged regions of molecules. Stealing protons from metal is not doable as the protons are buried deep in the atomic nuclei. As a consequence, bases don’t dissolve metals but can still attack organic matter where protons are easier to steal and other reactions can occur. That is to say, bases can still be used for corpse disposal [3].
Protein folding – the second part of the puzzle
Alright, so pH is an upside-down measure of the number of loose protons and acids are full of them. The free protons love stealing electrons that aren’t theirs which explains their reactivity. But where does tofu come into the picture? To understand the connection between pH and tofu, we have to take a look at proteins and how they are made.
I’m pretty confident that everyone knows that all cells in the bodies of all living things contain DNA and that DNA contains our genes. The genes in turn are different for different species and individuals and determine all sorts of things about us, like our looks, how effectively we digest starch and thousands of other things.
But just how do genes do that? How do you go from DNA to eye color?
The connection between genes and our bodies is a very complex topic, with lots of answers and intense, ongoing research. The short answer is that the genes are blueprints for our proteins. They contain the information for every single protein in your body.
Our genes are our very own blueprints for how to build our proteins. But the blueprint is just one part of the problem of how to make you you. It is like giving a contractor a stack of blueprints for a chair, a door, a window, a wall, and all other items a house contains and hope that they can figure out how many windows and chairs to build and where to put them to give you your dreamhouse.
While genes are the blueprints for our proteins, we also have complex machinery which regulates which genes are ‘turned on’ in which cell and when, how many proteins are to be produced, and a whole range of other things.
A long string of amino acids
Our genes contain blueprints for our proteins but how is this information stored and how is it turned into proteins?
Each protein is made up of many amino acids. There are quite a few different amino acids but, as the figure above shows, they all share a common backbone unit with an amine group in one end and an acid group in the other (hence the name). When they are joined together, the amine group of one will bind to the acid group of the other, creating a peptide bond.
Each gene in DNA contains the information of which amino acids should be used and in which order they should go. This information is copied from DNA to a temporary molecule called messenger RNA. This is sort of like printing a blueprint from a hard drive – the hard drive (DNA) keeps the original blueprint while the printed copy (messenger RNA) can be used on your construction site a few times before it is ruined.
The messenger RNA is then read by a molecular machine called a ribosome which grabs amino acids as specified by the RNA sequence. The amino acids are added to a long, growing chain – the protein to be. This is an incredibly complex and fascinating process that doesn’t cease to amaze me. Just the fact that this works and that we have figured out as much as we have about it is just astonishing.
A crumpled up necklace
Putting the different amino acids in the correct order is just the start. If this was the end result, all proteins would look quite similar (long ropes floating around) and work in largely the same way. Too boring and inflexible. While all amino acids share a common backbone, they have different side groups which give them their unique functionality. Some side groups can create strong bonds with each other (covalent bonds, disulfide bridges) while others are strongly attracted to each other, similar to the attraction water molecules feel toward one another (hydrogen bonds).
To get a better idea of how this works, it is useful to imagine the long chain of amino acids as a necklace with many different beads. Some beads are like magnets and attract each other, some other beads have Velcro stuck to them and can bind to each other (but not to the magnets). Some are big and bulky while others are hydrophobic (water-fearing) or hydrophilic (water-loving). (Just like oil ‘fears’ water and you can’t mix the two.) If the bead necklace is free to twist and turn, these different bead properties will cause the necklace to curl up in a way that lets the magnets and the Velcro pieces pair up while placing the water-loving beads on the outside and the water-fearing beads in the center, safe from the scary water. This highly controlled crumpling is called protein folding and creates the full 3D structure of the finished protein.
And that’s how the body takes information stored in DNA and converts it into a protein, such as the pigmentation in your eyes.
What about tofu?
Alright, so now we know how amino acids are like functional beads on a necklace and how many different interactions can create well-controlled, complex 3D structures. Still no tofu.
One of the important effects in protein folding is the water-fearing and water-loving amino acids/necklace beads. And this is where pH comes in. The amino acids that do like water and are facing outward are often charged. Adding a bunch of free protons will neutralize the charge on these amino acids, causing them to like water a lot less. And many of the previously uncharged amino acids in the core can become charged and start to be more attracted to water. This addition of protons is called protonation and also affects the amino acids’ abilities to attract each other (the necklace beads can lose or gain the ‘Velcro function’, the hydrogen bonding). These charge changes wreak havoc on which amino acids should be on the inside and which should be on the outside and parts of the protein flip inside out. This is called denaturation (the protein loses its natural shape – it is de-natured) [4], [5].
Reducing pH is just one of many ways to denature proteins – you can also replace the water with another solvent, heat the protein, or add chemicals like formaldehyde which creates bonds between nearby proteins. By denaturing proteins, the freshly exposed parts start binding to other proteins in an effort to hide their hydrophobic surfaces from the scary water surrounding them. They clump up in a process called coagulation or curdling.
Here’s tofu!
So – reducing pH leads to amino acids changing their charge which disrupts one of the mechanisms by which proteins maintain their shape. This disruption causes proteins to start clumping together – to coagulate. By now, I’m sure you see how we are getting to tofu, just like I promised.
Tofu is simply an application of this mechanism – take a water solution of protein, such as soy milk, add protons and watch as the proteins flip out and clump up. The soy milk curdles and that mass is mostly protein. Strain it out and you have tofu.
A simple experiment
In the photo above, you can see an experiment where I mixed acetic acid (5% acetic acid in water, sold as white vinegar) with store-bought soymilk. When adding a small amount of soy milk (pH 8) to vinegar (pH 3), the proteins instantly curdled up, creating thin strands of protein in a clear solution. When I instead added vinegar in small amounts to soymilk at room temperature, I did not get this extreme flip. Rather, the coagulation occurred more slowly and to a smaller extent, causing the soymilk to thicken to the texture of buttermilk. Even though I added 1/3 vinegar by volume (100 mL vinegar, 200 mL soymilk), I did not get the full curdling required for tofu.
Heat helps
Instead of adding copious amounts of vinegar to get tofu, tofu recipes use another trick. By heating the soymilk the proteins start to destabilize and it is easier to cause them to coagulate by reducing pH. To test this, I heated soymilk to 70C and then added a small amount of vinegar. I could instantly observe small protein blobs forming. (No coagulation occurred from heat alone.) When adding a little more vinegar, the soymilk thickened a lot more, quickly becoming much thicker than the room temperature experiment. When stirring, the blob of soymilk would move as a single body. Further time at high temperature will complete the coagulation and you will have soft tofu. Press out the water and you get firm tofu.
In cooking, people usually use lemon juice instead of vinegar but the principle is the same [6], [7]. The vinegar I used here had a pH of 3, the same as lemon juice. Often, you will also see recipes using different salts instead of acids, see sidebar [8].
The crudling of heated protein solutions in acidic environments is also responsible for some non-dairy milks curdling when added to coffee. The coffee is both warm and acidic.
Conclusion
There you have it. Proteins are like long necklaces where the individual properties of the beads determine the proteins 3D structure and its function. Low pH is nothing but a surpplus of protons and these wreak havoc on the different beads’ properties, causing the proteins to unfold and stick together. To help this unfolding, tofu recipes call for heating as well as adding protons.
I hope you got this far, learned something, and enjoyed reading this piece on vegan food chemistry, biology, and physics.
If you’ve read a few tofu recipes, you have probably seen that many don’t use acid but instead use salts with 2+ ions (calcium and magnesium salts are popular) . Causing proteins to coagulate using these salts works great but through a different mechanism.
One reason proteins don’t bunch up normally is that they are charged and repel each other.
By introducing ions with a 2+ charge to the water, you can ‘screen’ the charge from the proteins and the repulsion is reduced (like a molecular smoke screen) and the proteins can approach each other and clump up.
References
- [1]C. E. Ophardt, “pH scale,” Virtual Chembook – Elmhurst College, 2003. [Online]. Available: http://chemistry.elmhurst.edu/vchembook/184ph.html. [Accessed: 15-Jun-2020]
- [2]S. Sørensen, “Enzymstudien II: über die Messung und die Bedeutung der Wasserstoffionenkonzentration bei enzymatischen Prozessen,” Biochemische Zeitschrift, vol. 21, pp. 131–304, 1909.
- [3]R. Burks, “Can acid dissolve a body,” Chemistry World, 19-Jun-2017. [Online]. Available: https://www.chemistryworld.com/opinion/can-acid-dissolve-a-body/3007496.article. [Accessed: 12-Jun-2020]
- [4]T. A. Holme, “Denaturation,” Chemistry Explained. [Online]. Available: http://www.chemistryexplained.com/Co-Di/Denaturation.html. [Accessed: 14-Jun-2020]
- [5]L. Konermann, “Protein Unfolding and Denaturants,” eLS. John Wiley & Sons, Ltd: Chichester, May 2012, doi: 10.1002/9780470015902.a0003004.pub2. [Online]. Available: https://onlinelibrary.wiley.com/doi/full/10.1002/9780470015902.a0003004.pub2
- [6]M., “How to make Silken Tofu with soy beans and lemon juice,” mary’s test kitchen, 05-Apr-2018. [Online]. Available: http://www.marystestkitchen.com/diy-silken-tofu-soy-beans-lemon-juice/. [Accessed: 14-Jun-2020]
- [7]mikeinternet, “How to make tofu,” Instructables cooking. [Online]. Available: https://www.instructables.com/id/How-to-Make-Tofu/. [Accessed: 14-Jun-2020]
- [8]A. Nguyen, “How to make homemade tofu,” The Splendid Table, 02-Mar-2012. [Online]. Available: https://www.splendidtable.org/story/how-make-homemade-tofu. [Accessed: 14-Jun-2020]
2 thoughts on “Protons, proteins and tofu – vegan food chemistry”
What an amazing article! Super interesting and easy to follow despite my soft tofu brain.
Great to hear! Glad you could add some new info to your tofu