Rating antifreeze proteins: Not a breeze

Abstract

Antifreeze proteins (AFPs) are an interesting class of biomolecules that hinder macroscopic freezing by binding to small ice crystals and blocking their further growth. They were first discovered in the 1960s in arctic fish (1) and have since been observed in a wide range of other organisms, e.g., bacteria (2), fungi (3), plants (4), insects (5), and vertebrates (1,6), that live in low-temperature environments. AFPs are also of considerable practical interest due to their potential applications in cryopreservation (7), food processing (8), and hydrate inhibition (9). The most widely used metric for characterizing the antifreeze activity of an AFP is a quantity known as thermal hysteresis (TH) activity, which is typically determined from nanoliter cryoscopy experiments. In PNAS, Olijve et al. (10) demonstrate that the notion of the antifreeze potency of an AFP is complex and process-dependent, and using a single quantity such as TH activity measured from a particular assay is inadequate in predicting the performance of an AFP under different temperature regimes and processing conditions.To understand the origin of this complexity, a distinction must be made between traditional antifreeze agents (AFAs), such as salt and ethylene glycol, and AFPs. Traditional AFAs work by reducing Δμ, the thermodynamic driving force for crystallization. This leads to an effective depression in the equilibrium melting temperature, T f ,eq , proportional to the molal concentration (activity) of the AFA. In contrast, the antifreeze function of an AFP has a purely kinetic origin, emanating from its preferential binding to certain crystallographic planes of the (small) ice crystallites that might exist in the system. This hinders the nucleation and/or growth of such thermodynamically favored ice crystals, and henceforth results in a delay in macroscopic freezing. In this context, the TH of an AFP is defined as the difference between the equilibrium melting temperature and the kinetic freezing temperature of an ice crystal in its presence. This kinetic depression in freezing is typically orders of magnitude larger than the thermodynamic depression emanating from the dissolution of AFP molecules. This makes AFPs such an interesting class of macromolecules and enables them to operate at concentrations that are orders of magnitude smaller than what is needed for conventional AFAs.By construction, TH is a nonequilibrium quantity that not only depends on AFP concentration but also on other processing factors, such as the cooling rate and the crystallization stimulus (i.e., homogeneous, heterogeneous, or field-induced nucleation) used in a particular experimental procedure. The conventional method of choice for measuring TH is nanoliter cryoscopy (11), which is a two-step process. In the initial preparatory step, a small (∼ 1 nL) volume of the AFP-containing solution is flash-frozen either through rapidly cooling it to −40°C, or by using a milder quench in conjunction with an external stimulus (e.g., the application of mechanical excitation, ...

From ice-binding proteins to bio-inspired antifreeze materials

“…As TH activity is concentration dependent, 77 ζ = (hMP – hFP)/√ C AF(G)P has been introduced as a quantitative measure for TH activity. 66 , 78 Maximal TH activities measured by cryoscopy range from 0.1–0.5 °C in apoplastic extracts from overwintering plants 4 , 61 and ∼2 °C for 30–40 g L –1 protein in fish serum 21 , 74 to ∼2 to 13 °C for 10-times lower concentrations in insect hemolymph. 54 TH activity can be improved upon addition of (sub)molar concentrations of small molecular solutes 79 and ions, 20 , 80 conjugation 81 to or complexation 82 with potentiating proteins 82 or other ice-binding proteins 81 and polymers, 83 , 84 and IBS enlargement.…”

Computational investigation of surface freezing in a molecular model of water

“…F reezing of water into ice is ubiquitous in nature and can occur in a wide range of environments, from biological cells (1) and soil (2) to meteors (3) and atmospheric clouds (4). Ice formation is particularly important in the atmospheric processes that exert a major influence on our weather and climate.…”

Ice Binding Proteins: Diverse Biological Roles and Applications in Different Types of Industry

“…The most important factor is the specific arrangement of especially threonines. Further studies [89][90][91] on this issue showed more than two planes of ice that IBPs may bind to, and these were basal, primary prism, secondary prism, and pyramidal prism. The plurality of these planes caused different values of TH activity measured by cryoscopy and sonocrystallization in six examined AFPs of various origins bound to the ice faces specific for them.…”