Acidity of Frozen Solutions and Its Connection to Degradation of Enzymes during Freezing


                              Ľubica Krausková ^1, Dominik Heger ^1,2


^1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno,
Czech Republic

^2 Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University,
Kamenice 5/A29, 625 00 Brno, Czech Republic


Abstract

Constant pH is often essential for stability of many biomolecules. In solution buffers are used to
maintain pH value constant. However, when an aqueous solution of a salt freezes, pH of unfrozen
portion of the solution can change dramatically whether towards acidic or towards alkaline values,
even if a buffer is used. During freezing the solution becomes more concentrated because compounds
dissolved in the solution are expelled from a lattice of newly growing ice. This
freeze-concentration often results in excessive crystallization of less soluble component of the
buffer and more pronounced acidity changes than in a buffer-free solution. Such sequential buffer
crystallization was shown e.g. for sodium phosphate and succinate buffer systems.^1, 2
Freezing-induced acidity change was observed also in solutions of salts due to different
incorporation of anions and cations into the ice lattice and consequent neutralization of a arose
charge imbalance by the diffusion of the H^+ or OH^− from liquid into the ice.^3, 4 In this study
the effect of freezing-induced pH change on denaturation of model enzyme DbjA was examined.
Freezing-induced pH change was monitored for various experimental conditions and correlated to loss
of enzymatic activity during freezing and thawing.


References

1.            Murase, N.; Franks, F., Salt Precipitation During the Freeze-Concentration of
Phosphate Buffer Solutions. Biophysical Chemistry 1989, 34, (3), 293-300.

2.            Sundaramurthi, P.; Shalaev, E.; Suryanarayanan, R., Calorimetric and Diffractometric
Evidence for the Sequential Crystallization of Buffer Components and the Consequential Ph Swing in
Frozen Solutions. Journal of Physical Chemistry B 2010, 114, (14), 4915-4923.

3.            Workman, E. J.; Reynolds, S. E., Electrical Phenomena Occurring During the Freezing
of Dilute Aqueous Solutions and Their Possible Relationship to Thunderstorm Electricity. Physical
Review 1950, 78, (3), 254.

4.            Bronshteyn, V. L.; Chernov, A. A., Freezing Potentials Arising on Solidification of
Dilute Aqueous-Solutions of Electrolytes. Journal of Crystal Growth 1991, 112, (1), 129-145.