title: Suppression of protein inactivation during freezing by minimizing pH changes using ionic cryoprotectants.
abstract: Freezing and lyophilization are often used for stabilization of biomolecules; however, this sometimes results in partial degradation and loss of biological function in these molecules. In this study we examined the effect of freezing-induced acidity changes on denaturation of the model enzyme haloalkane dehalogenase under various experimental conditions. The effective local pH of frozen solutions is shown to be the key causal factor in protein stability. To preserve the activity of frozen-thawed enzymes, acidity changes were prevented by the addition of an ionic cryoprotectant, a compound which counteracts pH changes during freezing due to selective incorporation of its ions into the ice. This approach resulted in complete recovery of enzyme activity after multiple freeze-thaw cycles. We propose the utilization of ionic cryoprotectants as a new and effective cryopreservation method in research laboratories as well as in industrial processes.
authors.affiliation: Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno, Czech Republic; Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A29, 625 00 Brno, Czech Republic.
authors.forename: Ľubica
authors.initials: Ľ
authors.lastname: Krausková
authors.affiliation: Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno, Czech Republic.
authors.forename: Jitka
authors.initials: J
authors.lastname: Procházková
authors.affiliation: Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Klašková
authors.affiliation: Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Filipová
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic; Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A29, 625 00 Brno, Czech Republic.
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupková
authors.affiliation: Central Institute for Supervising and Testing in Agriculture, Hroznová 2, CZ-656 06, Czech Republic.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Malý
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic; Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A29, 625 00 Brno, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Damborský
authors.affiliation: Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A8, 625 00 Brno, Czech Republic; Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A29, 625 00 Brno, Czech Republic. Electronic address: hegerd@chemi.muni.cz.
authors.forename: Dominik
authors.initials: D
authors.lastname: Heger
mesh_terms: D002021:Buffers; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry
chemical_list: D002021:Buffers; D003451:Cryoprotective Agents; D007477:Ions; D011506:Proteins; D012996:Solutions
keywords: Freezing stress; Freezing-induced pH shift; Freezing/thawing; Protein aggregation; Protein cryopreservation; Protein stability; Quality by design