Cryopreservation of spinach chloroplast membranes by low-molecular-weight carbohydrates. II. Discrimination between colligative and noncolligative protection.

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of various concentrations of sugars, polyhydric alcohols, and NaCl. Functional integrity of the membranes was assayed by means of cyclic photophosphorylation. From the nonideal activity-concentration profiles of the carbohydrates the effective NaCl concentrations in the surroundings of the membranes at the respective freezing temperatures were calculated. Comparison of the cryoprotective efficiency of the various polyols revealed that cryopreservation by low-molecular-weight compounds is predominantly due to colligative action of the solutes. In addition, specific effects of carbohydrates which cannot be explained by the colligative concept are involved in cryoprotection. At NaCl concentrations exceeding 15 mm, the relative contribution of noncolligative membrane protection of a given polyol to overall cryopreservation was independent of the salt concentration. However, during freezing in the presence of very low salt concentrations, for instance 1-4 mm NaCl, cryoprotection due to colligative phenomena is reduced in favor of other mechanisms.