Chain elongation of diacylphosphatidylcholine induces fully bilayer interdigitation under atmospheric pressure

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Abstract

The phase transitions of dibehenoylphosphatidylcholine (C22PC) bilayer membrane were observed by differential scanning calorimetry under atmospheric pressure and light–transmittance measurements under high pressure. The constructed temperature–pressure phase diagram suggests that the gel phase at low temperatures is the interdigitated gel phase. To confirm the phase state, we performed small-angle neutron scattering and fluorescence measurements using a polarity-sensitive probe Prodan for the C22PC bilayer membrane under atmospheric pressure. The peaks obtained in both measurements clearly showed the characteristic patterns of the fully interdigitated gel phase. Taking into account of previous studies on the gel phase for long-chain PC bilayers under atmospheric pressure and our studies on the pressure-induced bilayer interdigitaion of diacyl-PCs, it turned out that the interdigitation of diacyl-PC bilayer membranes occurs when the carbon number of acyl chain reaches at least 22. The present study revealed that the interdigitation of PC bilayer membranes occurs not only by weakening the attractive force of polar head groups but also by strengthening the cohesive force of acyl chains. When dominating the force of acyl chains, the interdigitation can be induced even in a diacyl-PC bilayer membrane by only hydration under atmospheric pressure.

Graphical abstract

Schematic illustration of fully bilayer interdigitation of ester-linked dibehenoylphosphatidylcholine bilayer in water under atmospheric pressure.

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Research highlights

▶ C22PC bilayer membrane induces fully interdigitation under atmospheric pressure. Interdigitation is dominated by chain interaction as well as head-group interaction. The fact is consistent with the expectation from chain-length dependence of MIP. Chain elongation of diacyl-PC produces bilayer interdigitation by only hydration.

Introduction

Lipid bilayer membranes of diacylphosphatidylcholines (diacyl-PCs), of which two identical saturated fatty-acid chains are bound to the glycerol backbone by ester linkage, have been thoroughly examined by several physico-chemical techniques [1], [2], [3], [4], [5], [6], [7], [8], [9]. Such bilayer membranes form various kinds of phases depending upon experimental conditions, and the characteristic gel-phase polymorphism is observed. The fully interdigitated gel (LβI) phase is unusual gel phase where one side of monolayers constituting bilayers mutually interpenetrate the other side of monolayers. The LβI phase of the diacyl-PC bilayer membrane is usually induced by adding amphiphiles such as polyols, short-chain alcohols and anesthetics [10]. These inducers weaken attractive force between polar head groups, resulting in the interdigitated structure due to the van der Waals forces between the acyl chains. Without the additives, the LβI phase of the diacyl-PC bilayer membrane is also induced by applying hydrostatic pressure. Although the mechanism of the pressure-induced interdigitation for diacyl-PC bilayer membranes has not yet been clear, we speculate that the bilayer interdigitation may be caused by changing from the conventional attractive interaction between neighboring polar head groups to the transient repulsive interaction, which is produced by the transitory perpendicular conformation of the head groups under high pressure [11], [12], [13].

On the other hand, it is known that bilayer membranes of dialkylphosphatidylcholines (dialkyl-PCs), of which two identical alkyl chains are bound to the glycerol backbone by ether linkage, can form the LβI phase by only hydration under atmospheric pressure. This is attributable to the fact that the attractive interaction among the head group of dialkyl-PC molecules in the bilayer membranes is weaker than that of diacyl-PC ones, which is originated from ether bonds in the backbone [14], [15]. We have studied the effect of acyl-chain length on the bilayer phase behavior of a homologue of diacyl-PCs under high pressure systematically and revealed that the phase behavior is obviously influenced by the acyl-chain lengths of the molecule [16], especially the bilayer interdigitarion. A minimum interdigitation pressure (MIP), which is the minimum pressure required for the interdigitation, decreases with an elongation of acyl chains by the enhanced cohesive interaction between acyl chains of the molecule [11], [16]. This finding signifies that the interdigitation of diacyl-PC bilayer membranes occurs not only by weakening the attractive force of polar head groups, but also by strengthening the cohesive force of acyl chains. And then, there is a high possibility of the bilayer interdigitation for diacyl-PCs with longer chains under atmospheric pressure.

In fact, Sun et al. have studied bilayer phase behavior of diacyl-PCs with longer acyl chains under atmospheric pressure by X-ray scattering [17]. They demonstrated that a dilignoceroyl-PC (C24PC) bilayer induces a new phase at low temperatures and supposed that the phase would be the untilted interdigitated phase, not the hydrated crystal (sub-gel) phase. Although they referred to the similar behavior to a dibehenoyl-PC (C22PC) bilayer, the result was not enough to verify it. Furthermore, other researches have not shown [18], [19] that the C22PC bilayer is induced the LβI phase under atmospheric pressure. Therefore, the interdigitation for diacyl-PCs with longer chains under atmospheric pressure has not yet been clarified and further researches are required. In the present study, we investigate the bilayer interdigitation of C22PC under atmospheric pressure by using macroscopic and microscopic techniques and applying our previous studies with respect to diacyl- and dialkyl-PC bilayers under high pressure. The temperature (T)–pressure (p) phase diagram of the C22PC bilayer membrane is constructed by differential scanning calorimetry (DSC) under atmospheric pressure and light–transmittance measurements under high pressure and the phase diagram is discussed by comparing it with those obtained for bilayer membranes of diacyl- and dialkyl-PCs. Then, in order to identify for the low-temperature gel phase of the C22PC bilayer membrane, the phase assignment is carried out by small-angle neutron scattering (SANS) and Prodan fluorescence techniques under atmospheric pressure.

Section snippets

Materials and sample preparation

A synthetic phospholipid, 1,2-dibehenoyl-sn-glycero-3-phosphocholine (Lot# 83F-8400), was purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. Water was distilled twice from a dilute alkaline permanganate solution. The phospholipid multilamellar vesicles were prepared by suspending a phospholipid in water at 1.0 mmol kg−1 for DSC and light–transmittance measurements and in D2O at 10 mmol kg−1 for SANS experiments, respectively. The suspensions were sonicated for a few minutes by

Results and discussion

The DSC thermogram of the C22PC bilayer membrane is depicted in Fig. 1. We observed one sharp endothermic peak at 73.4 °C with a small shoulder peak in the front of the main peak. The enthalpy change (ΔH) of the total peaks was 65.4 kJ mol−1, which was consistent with the previous report [18]. From the systematic DSC measurements performed by us [16], [22] and Lewis et al. [18] previously, it was thought that a small shoulder peak corresponds to the pretransition from the lamellar gel (Lβ′) phase

References (25)

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