A Closer Look: TCA Protein Preps

Last week, Amy (our sensational PI) and Esther (who, along with Tati and Rozi, has now absconded for home, leaving me to fend for myself in Mac Lab) came to me with a quandary-slash-question and I was pleased when I realized that, with the help of some chemical structures and pKa values, I could answer it! Amy was super excited at the structures that I drew on our whiteboard and suggested of her OWN volition (FOR ONCE) that I blog about it! So here, for you now, is my own understanding of how TCA protein preps work on the chemical level.

Some background. Esther has spent the summer working with proteins (whereas I have been focusing on genetics and cytology, and Rozi and Tati's project involved a genetic screen) - specifically, she was investigating whether proteins involved in regulation of the assembly of the synaptonemal complex (SC) in meiotic chromosomes are themselves regulated by posttranslational modifications that are dependent on recombination. Yes, that is a mouthful - to rephrase it, what she would like to find out is Spo11, the major protein player in recombination that catalyzes double-stranded breaks in DNA to initiate recombination events, also has an effect on the regulation of SC-assembling proteins by having upstream control over if and when these proteins are modified (i.e. by SUMOylation), if at all, as the modification of these proteins may be dependent on the occurrence of recombination events. So for example, if the SPO11 gene was knocked out in a strain, would there be any effect on the physical state (covalent modifications) of SC-assembling proteins - and if so, would this suggest that a lack of recombination sites on the chromosomes led to the SC-assembling-protein posttranslational modification pathway not being activated?

To answer these questions she was using Western blotting and, EXCITINGLY, 2D gel electrophoresis to basically check out the size of different SC-related proteins - Fpr3, Zip2, Zip3, Zip4, and Rrd1 - in both SPO11+ and Δspo11 backgrounds to see if there was any difference in size of the SC proteins in the two different backgrounds. BUT OF COURSE, before she could run these proteins out on gels, she had to isolate them from cells of her strains. And how did she do that? BY TCA PROTEIN PRECIPITATION. This is where I come in.

The TCA protein prep protocol is a relatively straightforward one - it involves variations on the basic theme of pelleting yeast cells, vortexing the pellet with TCA and glass beads to lyse the cells and release the protein, and then washing with a basic resuspension solvent to force the total cell protein out of solution as a pellet. Cool, right? But what she wanted to know was HOW adding some TCA to cells actually precipitated ALL of the protein in the cell, with no regard to specificity or anything. She had read somewhere that "hydrophobic aggregation" was involved. So I looked up the structure and pKa of TCA, and a lightbulb TOTALLY went off.

TCA is trichloroacetic acid, a derivative of acetic acid:

(Yes, this post, like the zygote post, is going to be illustrated with my superlative Paint skills. At first I was using ChemSketch, a really awesome molecule-drawing freeware program from ADC/Labs that you can get here, but then I got tired of it because the molecules all looked so uniform. I think molecules should have PERSONALITY.)

In aqueous solution, TCA dissociates into its conjugate base plus hydrogen ions:

Note the pKa, 0.77. That is VERY LOW. As you will recall from biochem, orgo, or wherever you learned about the lovely world of dissociation reactions, pKa is a measure of the acidity of a molecule - more specifically, the equilibrium constant for the dissociation reaction, and a measure of a molecule's affinity for its acidic proton when in aqueous solution (water). Since pKa is a measure of the RATE of the dissociation, and in general, the rate, K, of a reaction is given by-

K = [reactants]/[products]

reactions with a very high K (and in turn a very low pK, because pK = 10^(-K)) have a very HIGH ratio of reactants to products in solution. In our case, this means that in solution, virtually all of the TCA has dissociated into protons and conjugate base, because the proton so readily dissociates from the hydroxyl group.

(Of course, we could take this down even another level and point out that TCA has such a low pKa because of the enormous electronegativity of the halogenated end of the molecule - chlorine is the second most electronegative atom out-electronegated only by fluorine, and THREE chlorines all on one end of the molecule STRONGLY polarizes the electrons in the molecule toward that end - this enormous pull for the electrons away from the hydroxyl group means that there is a very small net electron density on the hydroxyl group, not leaving much for the proton to "grip" onto so it pops off quite easily.)

But of course, I lied to you in that drawing above. Free H+ atoms NEVER exist in solution. They ALWAYS exist coordinated to water molecules by hydrogen bonds. So really, the above reaction looks like this:

Notice anything? Given the extremely low pKa of TCA, its rapid dissociation means that practically ALL of the water molecules in solution will be "occupied" by existing as hydronium (H3O+) ions!

Now let's zoom out to Esther's experiment. This is why I love biochem.

Esther's protocol called for adding TCA and glass beads to a cell suspension, vortexing vigorously, centrifuging and collecting the supernatant, and then pelleting out the protein from the super by resuspending in Tris base. NOW WE KNOW how the TCA makes the protein fall out of solution! Do you see it yet? (The glass beads and vortexing were simply physical trauma to the cells to lyse them and release the protein and other cell contents.)

When you add TCA to an aqueous solution, it dissociates IMMEDIATELY and the ENORMOUS concentration of protons intercalate with the water molecules, effectively blocking them from solubilizing the cellular protein (which is mostly hydrophilic, as most cellular proteins have at least one hydrophilic region). Since the TCA outcompetes the protein for hydrogen-bonding with water, the proteins have nothing to do but aggregate with themselves and fall out of solution as hydrophobic aggregations.

This picture is actually most accurate because each water molecule is hydrogen-bonding with two protons - because the oxygen in water has two lone pairs, each of which can coordinate one proton. Then when you spin this whole mixture at high RPMs, cell debris (like the cell wall and membrane and organelles) and protein fall completely out of solution, while water keeps the TCA busy (or vice versa, as the case may be) in the aqueous layer. Tossing out the aqueous layer yields a pellet containing all of your cellular protein! The pellet also contains unwanted cell debris, so a wash and spin in a very basic resuspension solution (i.e. Tris base) again forces the cell debris (which are not that polar) out of solution, FINALLY yielding a solution of all the cellular proteins.

Really cool chemistry behind a useful, (seemingly) straightforward protocol. :)