Re: Why is DNA resistant to phenol denaturation, yet proteins aren't?

Lisa,

Denaturation of proteins and purification of DNA by phenol are both solvation related phenomena. From this, we will see why phenol denatures proteins and not DNA. I'll answer your question about proteins first, then look at DNA.

Proteins

There is a well known saying that "like dissolves like". That is, polar solutes dissolve well into polar solvents and likewise for nonpolar materials, whereas nonpolars and polars do not mix well. This is because like-unlike mixing incurs a large thermodynamic penalty.

As an example, let's look at water as a solvent. The hydrogen-bonding properties of water molecules cause the formation of a cage-like structure in order to surround nonpolar solutes. This structure, called a clathrate, is relatively ordered and reflects a loss of entropy (disorder), which is thermodynamically unfavorable.

We can now use this property to explain, in general, how proteins fold. Proteins tend to fold so that hydrophobic side chains are in the interior, unexposed to water molecules. Hydrophilic side chains remain on the exterior. Otherwise, there would be a large thermodynamic penalty due to the entropic cost of water hydration. This is known as the hydrophobic effect.

However, if the solvent is changed to phenol, an organic hydrophobic, the thermodynamic penalty is eliminated. Hence, the entropy loss due to protein folding becomes the overriding factor, and protein unfolding (denaturing) is energetically advantageous. This is why phenol will denature proteins.

DNA

In a typical purification of DNA, phenol and chloroform are used to do a gross purification of DNA. Organics and proteins are removed in this extraction process. DNA is not soluble in these solvents, so it remains in the aqueous layer. Thus, addition of phenol should not denature DNA. The fact that DNA codes for proteins biologically has nothing to do with the chemistry behind its interaction with phenol.

Plus, you wouldn't expect denaturation anyway even with DNA dissolved in pure phenol. Phenol is hydrophobic and DNA is both charged (phosphate groups) and hydrogen bonds copiously (bases). So similar to the previous reasoning, DNA denaturation should be thermodynamically unfavorable.

As an aside, extraction is usually followed by the addition of salt and ethanol precipitation. By adding ethanol, the dielectric constant of the solvent is lowered. This means that long-range electrical forces become significant, causing the salt cations and the negatively charged DNA to agglomerate and precipitate out of solution.

Summary

So, the upshot of this is:

• Phenol denatures proteins because it solvates the hydrophobic interiors of proteins.
• Phenol does not denature DNA because phenol is hydrophobic and DNA is hydrophilic.

Thanks for the interesting question. Stay curious!

Your MAD Scientist,
Raymond Cheong

References:

Blaber, Michael. "Water and the hydrophobic effect" Fall 1995.

Williams, Loren. "Molecular Interactions." 30 Mar 1999.