Can life exist without water? This is one of the questions that fascinates astrobiologists. The behavior of biomolecules in non-aqueous solutions is also of interest to cryobiologists and cryoenzymologists. Ice formation below zero degrees Celsius can be prevented by high concentrations of cryoprotective agents. But what are the effects of such vitrification agents on proteins?

In 1989 Alexander M. Klibanov published a paper called “Enzymatic Catalysis in Anhydrous Organic Solvents” that reports that enzymes are not only able to function in anhydrous organic solvents, but that some display remarkable properties in such environments like enhanced storage stability, solvent-induced changes of enzyme stereoselectivity, molecular memory, and reactions that are normally inhibited in aqueous solutions.

Upon reading the paper it is clear that when the author speaks of anhydrous solvents it is not implied that enzymes do not require water at all:

“…the key question should be not whether, but how much, water is required for enzymatic activity. Clearly, the enzyme molecule cannot ‘see’ more than a monolayer or so of water around it. Therefore, if this layer of ‘essential’ water is  somehow localized and kept on the surface of the enzyme, then all the bulk water should be replaceable with organic solvents with no adverse effects on the enzyme.”

To assure enzymatic activity in organic solvents two rules must be followed. First, hydrophobic solvents are preferred.  The authors propose that hydrophilic solvents ‘strip’ the essential water from the enzymes, and thereby reduce or eliminate the activity of enzymes. Second, the enzymes to be used in organic solvents need to be lyophilized (freeze dried) from aqueous solutions  with the pH optimal for their activity. This last requirement reflects the phenomenon of “pH memory” in which the enzymes retain the ionization state they had at that pH in the aqueous solution during freeze-drying and in organic solvents.

As surprising as some of these findings may be, the requirement of bound water for enzymes to  function is still consistent with the orthodox view that life requires water. At best, such findings can explain the existence or preservation of life in low water environments.

For cryobiologists, such findings raise interesting questions. In 2004, Fahy, Wowk et al. speculate that one of the mechanisms of cryoprotectant toxicity may involve “reduced hydration of biomolecules.” Understanding how solvents, and the combination of solvents, affect the intracellular milieu and the hydration and stability of biomolecules, should contribute to the design of less toxic vitrification solutions. Such vitrification solutions can be optimized for the human brain to allow for real suspended animation and improved prospects of resuscitation of cryonics patients.