We have studied the effects of cholesterol on the thermotropic phase behavior of aqueous dispersions of a homologous series of linear saturated phosphatidylcholines, using high-sensitivity differential scanning calorimetry and an experimental protocol which ensures that broad, low-enthalpy phase transitions are accurately monitored. We find that the incorporation of small amounts of cholesterol progressively decreases the temperature and the enthalpy, but not the cooperativity, of the pretransition of all phosphatidylcholines exhibiting such a pretransition and that the pretransition is completely abolished at cholesterol concentrations above 5 mol % in all cases. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition of these phospholipid bilayers in both hydrocarbon chain length-dependent and hydrocarbon chain length-independent ways. At cholesterol concentrations of from 1 to 20-25 mol %, the DSC endotherms of all phosphatidylcholines studied consist of a superimposed sharp and broad component, the former ascribed to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains. The temperature and cooperativity of the sharp component are reduced only slightly and in a chain length-independent manner with increasing cholesterol concentration, an effect we ascribe to the colligative effect of the presence of small quantities of cholesterol at the domain boundaries. Moreover, the enthalpy of the sharp component decreases and becomes zero at 20-25 mol % cholesterol for all of the phosphatidylcholines examined. In contrast, the broad component exhibits a chain length-dependent shift in temperature and a chain length-dependent decrease in cooperativity, but a chain length-independent increase in enthalpy over this same range of cholesterol concentrations. Specifically, cholesterol incorporation progressively increases the phase transition temperature of the broad component in phosphatidylcholines having hydrocarbon chains of 16 or fewer carbon atoms, while decreasing the broad-component phase transition temperature in phosphatidylcholines having hydrocarbon chains of 18 or more carbon atoms. We attribute this behavior to the effects of hydrophobic mismatch between the cholesterol molecule and its host phosphatidylcholine bilayer [see Mouritsen, O. G., & Bloom, M. (1984) Biophys. J. 46,141-153] and propose that the best match between the effective length of the cholesterol molecule and the mean hydrophobic thickness of the phospholipid bilayers is obtained with the diheptadecanoylphosphatidylcholine molecule. Moreover, cholesterol decreases the cooperativity of the broad component more rapidly and to a greater extent in the shorter chain as compared to the longer chain phosphatidylcholines. At cholesterol concentrations above 20-25 mol %, the sharp component is abolished, and the broad component continues to manifest the chain length-dependent effects on the temperature and cooperativity described above. However, the enthalpy of the broad component decreases linearly and reaches zero at about 50 mol % cholesterol, regardless of the chain length of the phosphatidylcholine. This latter finding does not agree with a previous study [Singer, M. A., & Finegold, L. (1990) Biophys. J. 57, 153-156], which found that the cholesterol concentration required to reduce the enthalpy of the main phase transition to zero appeared to increase steeply and approximately linearly with phosphatidylcholine hydrocarbon chain length. We ascribe these previous results to an experimental artifact arising from the use of a low-sensitivity calorimeter and an experimental protocol not optimized to detect broad, low-enthalpy phase transitions.
