Volume 59 (2013) Issue 1 Pages 15-21
Antifreeze proteins (AFPs) are a subset of ice-binding proteins that evolved to arrest ice crystal growth and also inhibit the recrystallization of ice. An accepted theory for their activity, named the adsorption-inhibition hypothesis, posits that AFP binding to ice must be irreversible because the ice crystals do not grow within a freezing hysteresis (FH) gap. This theory has been criticized due to several experimental observations such as the dependence of FH values on the AFP concentration. Here we present recent fluorescence and microfluidics experimental results of our group supporting irreversible binding of AFPs to ice surfaces. i) Photo-bleaching of GFP-tagged AFP residing on the surface of an ice crystal held in the FH gap showed that there is neither exchange nor overgrowth of the bleached AFP. ii) Ice crystals bound by AFPs showed a measurable resistance to melting (melting hysteresis) demonstrating that the AFPs remain surface-bound at temperatures above the equilibrium melting point. iii) Using a temperature controlled microfluidic apparatus we demonstrated that small ice crystals formed in AFP solution stay intact even after the surrounding solution is depleted of AFPs. Additional measurements of surface fluorescence made during and after the exchange procedure showed no decrease in the fluorescence intensity, suggesting that bound AFPs did not leave the ice surface during the process. In light of these results, this review concludes that surface-adsorbed AFPs are the core source of the freezing hysteresis activity of the tested AFPs and that their adsorption to ice is irreversible.