Abstract
Antifreeze proteins (AFPs) protect organisms living in subzero environments from freezing injury, which render them potential applications for cryopreservation of living cells, organs, and tissues. Cryoprotective agents (CPAs), such as glycerol and propylene glycol, have been used as ingredients to treat cellular tissues and organs to prevent ice crystal’s formation at low temperatures. To assess AFP’s function in CPA solutions, we have the applied site-directed spin labeling technique to a Type I AFP. A two-step process to prevent bulk freezing of the CPA solutions was observed by the cryo-photo microscopy, i.e., (1) thermodynamic freezing point depression by the CPAs; and (2) inhibition to the growth of seed ice crystals by the AFP. Electron paramagnetic resonance (EPR) experiments were also carried out from room temperature to 97 K, and vice versa. The EPR results indicate that the spin labeled AFP bound to ice surfaces, and inhibit the growths of ice through the bulk freezing processes in the CPA solutions. The ice-surface bound AFP in the frozen matrices could also prevent the formation of large ice crystals during the melting processes of the solutions. Our study illustrates that AFPs can play an active role in CPA solutions for cryopreservation applications.
Highlights
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) protect organisms living in subzero environments from freezing injury, and even death [1,2,3,4,5,6,7,8,9,10]
After flash freezing the whole solution to a low temperature (
A seed ice crystal had to grow on the basal planes with the decrease in temperature due to the binding of AFP to the other facets, and its shape was confined to a bipyramid by the AFP while the rest of the solution stayed in super-cooled condition
Summary
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) protect organisms living in subzero environments from freezing injury, and even death [1,2,3,4,5,6,7,8,9,10]. It was reported that HPLC6 peptides bind to the 12 equivalent bipyramidal planes of ice Ih (hexagonal ice) along the direction [24]. The binding of the AFP to these surfaces confines the growth of ice crystal to a bipyramidal shape [25,26,27]. Many experimental results from the studies of mutagenesis and their antifreeze activities [27,28,29,30,31,32,33], solid-state NMR [17,18,19], and site-directed spin labeling technique [34] have demonstrated that the underlined residues in the above sequence bind to the ice surface
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