Interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles. II. A fluorescence polarization study.

The interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles was studied as a function of temperature, pH and the molar ratio of phospholipid to protein. The method consisted of measuring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene used as a probe embedded in the vesicles. After incubation of the protein with the phospholipid for 2 h at 23 degrees C, the polarization of the light emitted by this probe shifted to higher values; the shift was greater at acidic pH than at neutral pH. After incubation at 37 degrees C, no shift in polarization was found at pH 7, 6 and 5 while a strong increase occurred at pH 4. Lowering the temperature, after incubation at 37 degrees C, had little effect on the polarization at neutral pH. At pH 5, however, and in the transition range of the phospholipid, the polarization increased greatly. A kinetic study of the interaction carried out around the transition temperature of dimyristoyl phosphatidylcholine as a function of pH shows that the speed of complex formation between alpha-lactalbumin and the lipid increases from neutral to acidic pH. From the present results and in agreement with our earlier calorimetric and fluorescence data (Hanssens, I., Houthuys, C., Herreman, W. and van Cauwelaert, F.H. (1980) Biochim. Biophys, Acta 602, 539--557), it is concluded that at neutral pH the interaction mechanism is probably different from that at acidic pH. At neutral pH and at all temperatures, alpha-lactalbumin is mainly absorbed electrostatically to the outer surface of the vesicle with little or no influence on the transition temperature of the phospholipid. At this pH, only around the transition temperature is penetration possible. At pH 4, however, the protein is able to penetrate the vesicle at all temperatures and to interact hydrophobically with the phospholipid fatty acid chains. As a result of this interaction, the transition temperature is increased by about 4 degrees C. This different behaviour changes progressively upon acidification: at pH 5, penetration seems to be impossible at temperatures far above the transition temperature but occurs rapidly around the transition temperature.