Proteins were isolated from cold-acclimated cabbage leaves that protect isolated spinach thylakoids against freeze-thaw damage. Three different assay methods are described to quantitate the activity of cryoprotective proteins. Isolated spinach thylakoids were frozen in the presence of different amounts of protein. The membranes were either suspended in 2.5 mM NaCl and 5 mM sucrose or in a simplified chloroplast stroma medium to generate freeze-thaw damage during a 3 h incubation at -20-degrees-C. Damage and protection can be quantitated as the release of the soluble lumenal protein, plastocyanin, in both solute systems. Alternatively, in the presence of the NaCl/sucrose solution, rupture and concomitant collapse of the membrane vesicles can be measured as packed thylakoid volume by haematocrit centrifugation. This simple and inexpensive method was used to investigate the heat stability of cryoprotective proteins. These proteins were not precipitated by boiling. Their specific activity increased after an incubation at 70-degrees-C or more, due to the massive precipitation of contaminating proteins.
