Jan P. Wolanczyk

Ice nucleating activity in the blood of the freeze-tolerant frog, Rana sylvatica

Although the presence of antifreeze and ice nucleating agents in the hemolymph of insects has been well documented, there have been no reports of either of these types of agent in vertebrates. The technique of differential scanning calorimetry was used to examine the blood, serum, and plasma of a freeze-tolerant frog, Rana sylvatica, for the presence of antifreeze protein activity. Results demonstrate the absence of antifreeze protein but the presence of an ice nucleating agent that may serve as a functional component of the overwintering strategy of this species. Ice nucleating activity was detected in samples of cell-free blood, serum, and plasma, suggesting that the agent is a soluble component and possibly plasma protein. To our knowledge, the identification of ice nucleating activity in this freeze-tolerant vertebrate is novel.

Biochemical modification of plasma ice nucleating activity in a freeze-tolerant frog

Recently, we reported the presence of ice nucleating activity, apparently proteinaceous, in the plasma of a freeze-tolerant frog, Rana sylvatica, collected in autumn and spring. Although this protein has not been purified, its ice nucleating behavior can act as an internal reference for tests that attempt to modify its ability to nucleate ice formation. If the addition of a chemical reagent alters the temperature of ice crystallization compared with the control, it can be assumed that protein modification may have occurred. The ice nucleating protein in R. sylvatica showed resistance to proteolysis with four different proteases although there was a significant reduction in the temperatures of nucleation with these treatments (ANOVA P less than 0.001). However, ice nucleating activity was lost when plasma was treated with the addition of urea or N-bromosuccinimide. Modification of protein sulphydryl groups with iodoacetamide did not affect the crystallization temperature (Tc) but treatment with iodoacetic acid resulted in a significant increase in Tc of plasma. An abrupt loss of ice nucleating ability was observed in plasma samples after heating above 87 degrees C. Anomalous potentiation of ice nucleating activity occurred when the plasma was heated to and held at temperatures between 67-75 degrees C.