How to measure the activity of antifreeze proteins?

Investigation of a protein, such as an enzyme, requires an experimental method for assessing the activity of the protein, that is, some parameter related to its function. Determining protein activity is particularly important for understanding protein function under different conditions, as well as for designing new proteins or comparing proteins from different organisms. Antifreeze protein activity is a very specific type of activity and is very difficult to measure. One of the reasons is technical difficulties, since it is challenging to conduct experiments with aqueous solutions at sub-zero or near-zero temperatures. Another reason is that the mechanism of antifreeze proteins in different organisms is not fully understood, so the property most directly related to their primary physiological activity remains unclear. It is currently believed that antifreeze proteins may perform three functions: (1) influence the freezing point of water and melting point of ice, (2) affect the size of ice crystals or their growth process, and (3) inhibit ice nucleators within the cell or solution. Although all these functions are obviously related to each other, different methods are used to study them. These methods, aimed at assessing the various properties of antifreeze proteins, can be conditionally divided into three groups. In this paper, we reviewed the main methods for studying the activity of antifreeze proteins described in the literature.