Identifying Ice-Binding Proteins in Nature

Organisms inhabiting freezing terrestrial, polar, and alpine environments survive because they have evolved adaptations to tolerate sub-freezing temperatures. Among these adaptations are ice-binding proteins (IBPs) which in the case of fishes and some insects have antifreeze properties which allow them to avoid freezing even at their lowest environmental temperatures. Other organisms, including some insects, microorganisms, and plants, tolerate freezing and also contain IBPs. Unlike fish and insects, their antifreeze properties (hysteresis) are minimal, but most are potent ice recrystallization inhibitors (IRIs). Microbes secrete IBPs into their immediate environment where they are thought to modify ice growth in a way that ensures a liquidous habitat in the ice and also reduces ice recrystallization. With plants, IBPs are found in the small amount of apoplastic fluid associated with the extracellular spaces and show a weak hysteresis but are potent IRIs.Techniques are described for drawing blood and hemolymph from fish and insects, respectively, in order to determine whether there is a hysteresis present (separation of the freezing and melting points) indicative of an antifreeze protein. For microbes, which secrete very small amounts of IBPs into their environment, a technique is described where their spent growth media causes the pitting of the basal plane of an ice crystal at a temperature slightly below the media freezing point. In plants, IBPs are isolated from the apoplastic fluids of the leaves by vacuum infiltration of a fluid into the extracellular spaces and then recovering the fluid by centrifugation.The pitting of the basal plane again can be used to verify the presence of IBPs in the concentrated apoplastic fluid.The techniques describe how to collect fluids from a variety of organisms to determine if IBPs are present using nanoliter osmometry or using the ice basal plane pitting technique.