Differential scanning calorimetric analysis of Tenebrio molitor antifreeze protein activity

Abstract

Recently a new method for analysis of antifreeze proteins by differential scanning calorimetry has been developed (T. N. Hansen and J. G. Baust, Biochim. Biophys. Acta 957, 217–221). However, the parameters used were not examined for possible maximal activity. To test the parameters, pooled hemolymph samples of the common mealworm larva, Tenebrio molitor (25 °C, 16 hr:8 hr L:D), were collected and analyzed for activity. The samples were held at −40 °C and at various annealing temperatures for different lengths of time (0 to 360 min). No difference in activity was observed in the freeze intervals, while significant differences were observed in annealing times of less than 3 min. Hemolymph samples were also tested for antifreeze activity at various scan rates (0.1 to 10 °C · min −1). Significant differences in activity were observed for each rate. Both the short annealing times and the cooling rates were found to be due to methodology and not sample. The best parameters were found to consist of a 5-min freeze at −40 °C, a 5-min annealing interval, and a 1 °C · min −1 cooling rate. To test the optimized parameters, pooled samples of T. molitor hemolymph were monitored for changes in activity over time (up to 60 days) at various storage temperatures (−17, −80, −196 °C). No changes in activity were observed. These results suggest that care must be given to the reporting of the specific conditions used in the analysis of antifreeze activity.