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J. Phys. Chem. B 2000, 104, 10368-10378
Thermodynamics and Kinetics of the Early Steps of Solid-State Nucleation in the Fluid Lipid Bilayer Dmitri P. Kharakoz* and Elena A. Shlyapnikova Institute of Theoretical and Experimental Biophysics, RAS, 142292 Pushchino, Moscow, Russia ReceiVed: April 4, 2000
Temperature-scanning (10-70 °C) calorimetric, densitometric, and acoustic measurements have been performed in aqueous dispersions of dipalmitoylphosphatidylcholine multilamellar vesicles. In the biologically relevant liquid-crystalline state close to the chain freezing point (within 42-50 °C, in our case), the measured quantities display anomalous deviations from the “ideal” shape of their temperature dependence curves. The deviations indicate the early steps of gel-state nucleation within the stable liquid-crystalline state (Frenkel’s heterophase fluctuations). An earlier proposed kinetic model of nucleation (Kharakoz et al. J. Phys. Chem. 1993, 97, 9844-9851) has been refined to achieve more clear physical substantiation. When the deviations are analyzed through the refined model, both the thermodynamic and kinetic characteristics of nucleation are determined. The fluid-solid interface line tension energy is about 0.7 kT per boundary lipid molecule. The tension is strong enough for the phase transition to be first-order and, at the same time, weak enough to allow extensive heterophase fluctuations. The speed of new-phase-front propagation near the transition temperature is virtually proportional to the difference between the current temperature and transition temperature, the proportionality coefficient being 0.2 cm s-1 K-1 (estimated from ultrasonic relaxation data). The experimental data and the nucleation model are consistent with literature data on the anomalous “critical-like” behavior of lipid membranes near the main transition (bending rigidity drop, anomalous swelling, slowing relaxation processes, etc.). Thus, we show that most of the anomalies can be rationalized in terms of the weak first-order transition mechanism without a hypothesis on the proximity of bilayer to a critical state.
1. Introduction Much effort has been devoted to studying the mechanism of the liquid-crystalline to gel-phase transition in lipid bilayers, called the main transition.1-3 This phenomenon is interesting from both biological and physical points of view. Main transition evidently plays an important, although unclear, role in cell physiology.2,4,5 Furthermore, lipid bilayers in the biologically relevant statesa liquid crystal close to freezing pointsdisplay unusual physical behavior that has puzzled investigators. On one hand, the main transition behaves as a first-order transition characterized by pronounced discontinuity in the temperature dependencies of volume and enthalpy. The temperature interval of the transition completion is very narrow. In the case of multilamellar dipalmitoylphosphatidylcholine (DPPC), it is 0.07 K when accurately measured.5-7 On the other hand, the features commonly associated with second-order transitions are displayed. Specifically, within several degrees of the freezing point, extensive fluctuations of volume and in-plane area are detected. Consequently, the mechanical susceptibilitiessthe bulk and lateral compressibilitiessincreased pronouncedly, resulting in the anomalous behavior of related properties. The ultrasonic absorption is considerably increased and velocity reduced.8-14 The bending rigidity drops, and correspondingly, the undulations of the bilayer enlarge extensively.2,15 As a consequence, interlamellar repulsion forces increase,16 thus contributing to the anomalous swelling of multilamellar lipid systems.17,18 To rationalize the combination of the first-order character and the high fluctuations, the idea has been proposed that main * Corresponding author. E-mail:
[email protected].
transition should be considered a “pseudocritical phenomenon”,9,11,12,17 implying that a critical state, although not achieved, is close to the transition point. This idea, also supported by a “critical-like” slowing of relaxation processes, allowed consideration the anomalies on a phenomenological level in terms of the critical exponents. An alternative approach, initially intended to explain ultrasonic anomalies, has been proposed by Kharakoz et al.14 In their work (further referenced as work I), the anomalies were considered the indications of the so-called heterophase fluctuations. This term, suggested by Frenkel,19 implies the spontaneous creation and dissipation of the small, unstable nuclei of the new phase which the system approaches. A semiempirical kinetic model of nucleation developed in work I specifically for lipid bilayers agreed well with ultrasonic observations. The concept of heterophase fluctuations has provided a means for the quantitative analysis of the transition mechanism. For instance, the basic parameters governing the nucleation process have been evaluated for the first time for lipid bilayers: the line tension coefficient on the fluid-solid interface and the rate constant of an elementary event of nucleation. The former quantity can serve as a criterion to discriminate between the first- and second-order mechanisms; the latter determines the speed of new-phase propagation. However, the earlier model contained a number of poorly substantiated assumptions, of which the most incomprehensible was the cooperative coupling between lipid monolayers, which the authors assumed to match the model with the experimental data. This work is aimed at answering the following questions. Can the kinetic model of nucleation be established more firmly on
10.1021/jp001299a CCC: $19.00 © 2000 American Chemical Society Published on Web 10/14/2000
Solid-State Nucleation in Lipid Bilayer an experimental basis to avoid arbitrary assumptions? Is the model specifically applicable to only the ultrasonic observations, or it is more general, covering other physical properties as well? For this purpose, the temperature-scanning differential calorimetric, densitometric, and ultrasonic measurements have been performed in dispersions of multilamellar vesicles of DPPC over a wide range of temperatures, 10-70 °C. The earlier model has been refined to achieve clearer physical substantiation. (section 3). Applying the model to the experimental results, we show (section 5) that all three sets of datasdensitometric, calorimetric, and acousticsindicate heterophase fluctuations. They are quantitatively described within a unitary approach based on the refined kinetic model. Finally, we show (section 6) that the model is not only consistent with the listed physical properties, but it also explains other anomalies reported in the literature. Therefore, we have shown in this work that most of the anomalies called pseudocritical phenomena can be consistently explaned in terms of the first-order transition mechanism without having to apply critical-state terminology. 2. Materials and Methods 2.1. Materials. Synthetic dipalmitoylphosphatidylcholine was purchased from Avanti and used without further purification. The lipid powder was dried over 1 week under P2O5 in a vacuumed desiccator prior to use. Multilamellar vesicles were prepared by mixing the powder with double-distilled water, incubating the mixture for 30 min at 45 °C (above the maintransition temperature), and vortexing it periodically. The lipid concentration was determined from the weight to an accuracy of 1%. Prior to placing the sample into a measuring cell, we degassed it in a gastight syringe to reduce the probability of bubble formation. 2.2. Measuring Techniques. The temperature dependence of mass density was measured with a differential pair of the oscillating-tube densitometric cells DMA-602 (Anton Paar, Graz, Austria) in the temperature-scanning regime. The sensitivity of the cells was 10-6 g/cm3 and the sample volume 1 mL. The cell constants were determined by calibration with air and water in the range of temperatures studied. To accurately register the temperatures (see below), we registered the period of oscillations simultaneously in both cells. The standard electronic unit DMA-60 did not provide this opportunity. For this reason, we have designed our own electronic board to permanently monitor the periods in both cells simultaneously and to send the pairs of numbers to a personal computer for further processing. The precipitation of lipid vesicles creates serious obstacles to taking measurements because the oscillation of the measuring tube results in the uncontrolled redistribution of the sediment mass along the tube. To solve this problem and, at the same time, the problem of air bubble formation upon heating, we constructed a thermostated mixing-degassing vibropump, shown schematically in Figure 1. The pump (of 0.6 mL inner volume) provided permanent circulation of the sample through the measuring cell with a flow rate of about 10 mL/min, thus preventing sedimentation. The elastic elements of the pump are made of silicon tubes. Slight extra pressure applied to the inner space of the pump forces the dissolved gas to diffuse out of the sample through the silicon walls serving as semipermeable membranes. The inner concentration of air is therefore maintained below the saturation point. The extra pressure of 0.5 atm (stable within 2%) was enough to prevent bubble formation at any temperature and scan rate. The temperature in the densitometric cells and the thermostating jacket of the pump was changed with an external circulating water bath common for
J. Phys. Chem. B, Vol. 104, No. 44, 2000 10369
Figure 1. Schematic diagram of the mixing-degassing vibropump with heat exchanger. The three elastic elements of the pump (pulsating tube and two pulse dampers) are made of silicon, which is permeable for air. A slight extra pressure (0.5 atm) forces the dissolved air to flow out of the sample through the silicon walls.
both cells. Current temperature in the sample was calculated from the period of oscillation of the reference cell filled with pure water. The “period-temperature” relation has been preliminarily calibrated in the steady-temperature regime (stepby-step) with mercury thermometers accurate to (0.05 K. When measuring in the temperature-scanning mode, we took into account the systematic difference in the temperatures of the two cells (resulting from nonidentical heat exchange conditions due to the vibropump attached to the sample cell only). The slow mechanical relaxation of the DMA-602 cells was found to influence negligibly the determination of temperature. The final absolute error in temperature was
