Freezing the effect of eutectic crystallization on biological membranes

Thylakoids from isolated spinach chloroplasts were frozen in the presence of various concentrations of inorganic and organic salts, amino acids and sugars and the kinetics of inactivation of cyclic photophosphorylation with phenazine methosulfate and of electron transport reactions were measured as a function of temperature.During freezing of membranes in the presence of neutral nontoxic compounds membrane damage did not occur until the eutectic temperature was reached. Then photophosphorylation became rapidly inactivated. With weakly membrane-toxic compounds there was a slow inactivation during freezing followed by rapid inactivation at the eutectic temperature. Freezing in the presence of strongly membrane-toxic compounds led to inactivation of photophosphorylation before the eutectic temperature was reached. The temperature at which eutectic crystallization occurred was dependent on the nature of the solutes present. The ratio between solute and membranes was also important: the lower the initial concentration of solutes added to membrane suspensions the lower the temperature at which eutectic solidification occurred. Some compounds such as mannitol crystallized gradually during the decrease in temperature; in this case inactivation of photophosphorylation took place parallel to the crystallization process.In contrast to photophosphorylation, electron transport reactions were not decreased during eutectic freezing in the presence of neutral membrane-protective compounds. Rather a stimulation of electron transport was observed. However, in the presence of inorganic salts or of sodium succinate, electron transport reactions were also inactivated in addition to photophosphorylation during eutectic solidification. This inactivation seems to be a salt effect and may not directly be related to the crystallization process. Various soluble enzymes and the Ca²⁺-dependent ATPase of thylakoids were not affected by eutectic crystallization.The results demonstrate that eutectic crystallization which may take place during freezing is a factor in membrane damage and has to be considered as a possible cause of membrane alterations in in vitro studies on freezing resistance.