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Langmuir 2002, 18, 6716-6717
Comments Comment on “Trehalose Interacts with Phospholipid Polar Heads in Langmuir Monolayers”
In a recent paper, Lambruschini et al.1 have analyzed the interaction of trehalose with dipalmitoylphosphatidylcholine monolayers and its effect on the surface pressure and on the dipole potential. They concluded that trehalose interacts with the phospholipid polar heads and argued that the sugar participates in a network of hydrogen bonds between the phospholipid heads. They interpret their results, obtained by measuring pressure/area curves, in terms of the socalled water replacement hypothesis.2 One of the purposes of this comment is to call attention to several papers of our laboratory (not cited) in which direct experimental evidence of the interaction of trehalose with the carbonyls of the phospholipids in the polar headgroups, the amount of water displaced in that interaction, and the corresponding effect on the dipole potential was provided.3,4 The main results can be summarized as follows: 1. Trehalose interacts strongly in an excess of water with the carbonyls in the sn1 and sn2 positions of dimyristoylphosphatidylcholine (DMPC) as shown by a shift to lower frequencies in the Fourier transform infrared (FTIR) spectra. This shift denotes that the hydrogen bonding of trehalose with the carbonyl is stronger than those formed with water. 2. The amount of water displaced by trehalose in the above conditions is 60% of the total hydration level of DMPC. It may be derived that trehalose, at saturating conditions, displaces around 11 water molecules/phosphatidylcholine. 3. At those trehalose concentrations, the dipole potential of DMPC monolayers is decreased by around 100 mV. The FTIR results are direct evidence of a specific interaction of trehalose with defined chemical groups in the polar heads. Moreover, more recently we have shown that phosphate is another site of hydration in which the water can be displaced by trehalose or phloretin.5 As a consequence of this specific interaction of trehalose with the carbonyls, it is worthwhile to discuss the meaning * To whom correspondence should be addressed. Phone: 54 11 49648249 (ext 210). Fax: 54 11 4508 3653. E-mail: adisalvo@ ffyb.uba.edu.ar. (1) Lambruschini, C.; Relini, A.; Ridi, A.; Cordone, L.; Gliozzi, A. Langmuir 2000, 16, 5467. (2) Crowe, J.; Carpenter, J. F.; Crowe, L. M. Annu. Rev. Physiol. 1998, 60, 73. (3) Diaz, S.; Amalfa, F.; Biondi de Lopez, A. C.; Disalvo, E. A. Effect of water polarized at the carbonyl groups of phosphatidylcholines on the dipole potential of lipid bilayers. Langmuir 1999, 15 (15), 5179. (4) Luzardo, M del C.; Amalfa, F.; Nun˜ez, A. M.; Dı´az, S.; Biondi de Lopez, A. C.; Disalvo, E. A. Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers. Biophys. J. 2000, 78 (5), 2452. (5) Diaz, S.; Lairio´n, F.; Arroyo, J.; Biondi de Lopez, A. C.; Disalvo, E. A. Contribution of Phosphate Groups To The Dipole Potential of Dimyristoylphosphatidylcholine Membranes. Langmuir 2001, 17 (3), 852.
Figure 1. Dimyristoylphosphatidylcholine (squares) and ditetradecyl-glycero-phosphocholine (triangles) monolayers were spread on water (filled symbols) and on 0.05 M trehalose solution (empty symbols) at 30 °C.
of the network hydrogen formation in the model presented by Lambruschini et al. The carbonyls of the ester unions in the phosphatidylcholine are divided in two populations. One of them corresponds to the CdO dipoles oriented normally to the lipid interface, with the other one being parallel to the membrane plane. Thus, networks of hydrogen bonds can be formed in the plane of the membrane or extending normally to it toward the aqueous phase. FTIR experiments denoted that trehalose interacts with both populations. However, in terms of packing and surface pressure those carbonyls in the plane appear to be more relevant. In addition to these results, it has been demonstrated that trehalose can insert into the lipid phase upon dehydration and remains in the structure after rehydration producing expanded areas.6 This is congruent with the interpretation that trehalose interacts strongly with the carbonyls in the plane of the membrane. To get an insight on the relevance of the carbonyl groups in the formation of hydrogen bond networks with trehalose, we have done some assays in monolayers of DMPC and of di-tetradecyl-glycero-phosphocholine (DM(ether)PC), both obtained from Avanti Polar Lipids (Alabaster, AL) (Figure 1). DM(ether)PC is a lipid in which the fatty acid chains are bound to the glycerol by an ether union; thus, carbonyls are absent. We followed the change in the surface pressure upon subsequent addition of DMPC and DM(ether)PC to the interface of solutions without and with 0.05 M trehalose at constant area. In the presence of trehalose, the saturation point of the monolayer of DMPC is reached at a much higher lipid concentration than in water (5 nmol in comparison to 2.5 nmol at 30 °C) (Figure 1). This indicates that the area per lipid in water is much higher than in trehalose. That is to say that the hydration of the lipids gives place to a much higher excluded area than when it is displaced by trehalose. (6) Viera, L. I.; Alonso-Romanowski, S.; Borovyagin, V.; Feliz, M. R.; Disalvo, E. A. Properties of gel phase lipid-trehalose bilayers upon rehydration. Biochim. Biophys. Acta 1993, 1145 (1), 157.
10.1021/la011797q CCC: $22.00 © 2002 American Chemical Society Published on Web 07/19/2002
Comments
However, when DM(ether)PC is spread on a similar trehalose concentration at the same temperature, the amount of lipids to reach saturation is comparable to those of DMPC and DM(ether)PC without trehalose. This is a strong indication that the resulting packing in DMPC is a consequence of the interaction of trehalose with the carbonyls. This may be due to the strong hydrogen bonding of the sugar with the carbonyls as observed by FTIR and the subsequent displacement of water.4 The analysis and results shown above call attention to the fact that the interaction of trehalose with the polar heads must be defined more precisely considering the specific chemical groups present in the polar heads, their orientation with respect to the membrane, and the amount
Langmuir, Vol. 18, No. 17, 2002 6717
of water involved in the interaction of trehalose with each of them. E. A. Disalvo,* F. Lairion, F. Martini, and S. Diaz
Laboratorio de Fisicoquı´mica de Membranas Lipı´dicas, Ca´ tedra de Quı´mica General e Inorga´ nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956 2p 1113 Buenos Aires, Argentina Received December 12, 2001 In Final Form: February 20, 2002 LA011797Q
