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Dehydrins are a family of proteins associated with cell dehydration. Drought, salinity, and high and low temperature may cause water loss from cells. Cold-induced dehydrins have been reported in several species. P-80 is a cold-induced 80 kDa dehydrin in barley. This protein has the same apparent molecular mass as Dhn5, previously described for barley cv Himalaya. P-80 was localized in the vicinity of vascular cylinders and in the epidermis of leaves and stems. Both tissues have been reported to be sites of early ice nucleation during controlled freezing. The present authors have proposed that this protein cryoprotects macromolecules and frost-sensitive structures. In the present study, P-80 and Dhn5 were purified with the purposes of demonstrating their cryoprotective activity in vitro, and comparing both proteins. More than 95% purity was obtained combining heat treatment, cationic exchange chromatography, preparative denaturant electrophoresis and band electroelution. Western blots showed that P-80 was the major cold-induced dehydrin in the cultivars examined in the present study. There was a major band of mRNA that showed expression kinetics consistent with P-80 accumulation. The RT-PCR picked one major band when using Dhn5-specific primers in four cold-acclimated barley cultivars. Both proteins have a similar amino acid composition, with differences in Arg, Asn + Asp, Glu + Gln, His, and Lys. The analysis of proteolytic fragments of Dhn5 and P-80 by reverse phase chromatography showed a similar pattern. Furthermore, both proteins were able to cryoprotect lactate dehydrogenase (LDH, EC 1.1.1.27) against freeze/thaw inactivation, showing a similar shape dependence on concentration and almost the same protein dosage that renders 50% of cryoprotection (PD50). Thus, P-80 and Dhn-5 share more similarities than expected for two different proteins. Their identities, though, remain to be firmly established. Further research is necessary to establish if the observed in vitro cryoprotective activity of these dehydrins is important for cryoprotection in vivo. The association of cryoprotective activity with K repeats of dehydrins is discussed.