Protection mechanism of Tween 80 during freeze-thawing of a model protein, LDH.
The purpose of the study was to investigate the protective mechanism of a non-ionic surfactant, Tween 80, at freeze-thawing with controlled temperature history of a model protein, lactate dehydrogenase (LDH). The system was examined by differential scanning calorimetry (DSC) and infrared spectroscopy (IR). LDH activity assays were performed spectrophotometrically. In all samples, independent of temperature history and addition of surfactant, all water was crystallized to polycrystalline ice at temperatures below -20 degrees C. The size and perfection of the ice crystals could be varied by a range of cooling rates giving different degrees of undercooling. At Tween concentrations below the cmc at crystallization, lower concentrations were required at low cooling rates compared to higher cooling rates to protect LDH. Concentrations above cmc of Tween reduced the protection at a cooling rate of 5 degrees C min(-1) and at quenching in N(2)(l). The amount of Tween needed for complete protection correlated to the surface area of the ice crystals at a certain temperature history. Tween 80 protects LDH from denaturation at freeze-thawing by hindering its destructive interaction with the ice crystals. The protective effect might be obtained when Tween molecules compete with the protein for sites on the ice surface. The optimum concentration of Tween needed for complete protection is dependent on the temperature history.
Hillgren
Anna
A
Department of Pharmacy, Physical and Inorganic Pharmaceutical Chemistry, Uppsala University, Biomedical Center, P.O. Box 580, S-75123 Uppsala, Sweden.
Lindgren
Jan
J
Aldén
Maggie
M
D003080:Cold Temperature / Q000009:adverse effects; D005079:Excipients / Q000737:chemistry / Q000378:metabolism*; D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D011136:Polysorbates / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism
D005079:Excipients; D011136:Polysorbates; D011506:Proteins; D007770:L-Lactate Dehydrogenase