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Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 degrees C to 60 degrees C with Delta G degrees(321) of 13.9 +/- 0.3 kJ/mol and T-m value of 84 +/- 1 degrees C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular beta-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry. This is the peer-reviewed version of the following article: Rašković, B.; Popović, M.; Ostojić, S.; Ancrossed D Signelković, B.; Tešević, V.; Polović, N. Fourier Transform Infrared Spectroscopy Provides an Evidence of Papain Denaturation and Aggregation during Cold Storage. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2015, 150, 238–246. [https://doi.org/10.1016/j.saa.2015.05.061]

In winter flounder, the levels of type I antifreeze protein (AFP) and its mRNA vary seasonally by as much as 1000-fold. Elevated levels in the fall are prompted by the loss of long day-lengths, while higher spring temperatures correlate with AFP clearance. We have investigated the role of temperature on AFP accumulation using transgenic Drosophila melanogaster by expressing multiple AFP genes under control of the heat-inducible hsp70 promoter. AFP and AFP mRNA persisted far longer in flies reared at 10 degrees C compared to 22 degrees C. This difference appears to be mediated by cold-specific mRNA stability since no such temperature effect was observed with either an endogenous heat-inducible mRNA or a constitutively expressed mRNA.

Cold-stable and cold-labile microtubules were prepared by two cycles of assembly and disassembly and two periods of exposure to cold. The cold-labile preparations were shown to contain a higher proportion of a high molecular mass microtubule-associated protein (MAP 2) than cold-stable preparations. The cold-stable preparations showed a much higher alkaline phosphatase activity. Stimulation of microtubule assembly by zinc led to increases in both cold stability and alkaline phosphatase activity.