Oleate vesicles

The time course of an actual experiment is shown in Figure 7.17, which shows the hydrolysis of oleic anhydride catalyzed by spontaneously formed oleate vesicles. Note the sigmoid behavior, typical of an autocatalytic process. The lag phase is due to the preliminary formation of vesicles, and in fact the length of the lag phase is shortened when already formed vesicles are pre-added, as shown in the hg-ure. Some mechanistic details of these processes will be discussed in Chapter 10. In this work, an analysis of the number and size distribution of vesicles at the beginning and the end of the reaction was also performed by electron microscopy. 

Of all mentioned prebiotic membranogenic molecules, the ones that have gained more attention in the literature are long-chain fatty acids. In addition to their prebiotic relevance, these compounds are relatively simple from the structural point of view, and most of them are easily available. We will see in the next chapter that these vesicles have acquired a particular importance in the held of the origin of life. In fact, the hrst inveshgations on self-reproducing aqueous micelles and vesicles were carried out with caprylate (Bachmann et al, 1992) and most of the recent studies on vesicles involve vesicles from oleic acid/oleate (for simplicity we will refer to them as oleate vesicles). In this section, I would like to illustrate some of the basic properties of these surfactant aggregates. 

The cac (critical aggregate concentration) values for oleate are in the millimolar range, which means that at the operational concentration of 10-50 mM there will be a signihcant concentration of monomer in equilibrium with the aggregate. This consideration allows us to go back to the question of whether vesicles are chemical equilibrium systems. Oleate vesicles cannot be considered proper chemical equilibrium systems, however they behave in a mixed way, with some features that are typical of micelles in equilibrium (Luisi, 2001). 

Figure 10.7 Direct observation of transformations in giant vesicles. This results from the addition of oleic anhydride to giant oleate vesicles. (Adapted from Wick...

Let us consider the mechanism. When monomeric oleate or oleate micelles are added to a solution containing oleate vesicles, two limiting situations may occur. 

In contrast to this is the addition of oleate surfactant - in the form of micelles or free monomer - to oleate or to POPC vesicles. In this case, the ratio of the two competitive rates is such that a considerable binding of the added fresh surfactant to the pre-existing vesicles takes place. The efficient uptake of oleate molecules by POPC liposomes (Lonchin et al., 1999) as well as to oleate vesicles (Blochiger et al., 1998) is well documented in the literature. 

Figure 10.16 Effect of Ca + on the turbidity change upon mixing oleate vesicle solutions 0.25 ml 1 mM 60 nm radius extruded oleic acid vesicles + 0.25 ml 1 mM 200 nm radius extruded oleic acid vesicles + 1.5 ml bicine buffer. Calcium ion concentration (a) 0 mM (b) 1 irtM (c) 2.5 mM (d) 5 mM (e) added excess EDTA to (d). (Adapted from Cheng and Luisi, 2003.)...

One such surprise has been observed recently in a series of experiments in which negatively charged oleate vesicles were mixed with the positively charged DDAB... 

Figure 10.19 Effect of pre-added vesicles on the formation of oleic acid/oleate vesicles. Turbidity measured at 500 nm (1 cm path length) is plotted as a function of time, T=2°C. (a) 62 p,l of 80 mM aqueous sodium oleate was added to 2.438 ml of0.2Mbicine buffer, pH 8.8 ([oleic acid/oleate] = 2 mM). (b)62 p,l of 80mM aqueous sodium oleate was added to 2.438 ml of a 2 mM oleic acid/oleate 100 nm vesicle suspension (0.2 M bicine buffer, pH 8.8 ([oleic acid/oleate] = 4 mM). (c) Turbidity of 2 mM oleic acid/oleate 100 nm vesicles , (d) the same as (b), but using a 50 nm vesicle suspension, (e) Turbidity of 2 mM oleic acid/oleate 50 nm vesicles . (Modified from Blochiger etal, 1998.)...

Consider the experiment illustrated in Figure 10.19, which shows oleate vesicle formation when an aliquot of concentrated surfactant is added to water compared to the situation in which the same amount of surfactant is added to a solution containing pre-formed vesicles. In the second case, the formation of vesicles is remarkably accelerated, as if in the presence of a strong catalytic effect whereas over one hour is needed to reach the turbidity plateau for oleate addition to water, the plateau is reached in less than ten minutes, curve (b), in the second case. 

Figure 10.20 (a) Matrix effect for oleate addition to pre-formed POPC liposomes. In this case, mixed oleate/POPC vesicles are finally formed. Note the extraordinary similarity between the size distribution of the pre-formed liposomes and the final mixed ones. By contrast, the size distribution of the control (no pre-existing liposomes) is very broad, (i) Sodium oleate added to POPC liposomes, radius = 44.13, P-index = 0.06 (ii) POPC liposomes, radius = 49.63, P-index = 0.05 (iii) sodium oleate in buffer, radius = 199.43, P-index = 0.26. (b) matrix effect for the addition of fresh oleate to pre-existing extruded oleate vesicles. In this case, the average radius of the final vesicles is c. 10% greater than the pre-added ones, and again the difference with respect to the control experiment (no pre-added extruded vesicles) is striking, (i) Oleate vesicles extruded 100 nm, radius = 59.77, P-index = 0.06 (ii) oleate added to oleate vesicles, extended 100 nm, radius = 64.82, P-index 0.09 (iii) sodium oleate in buffer, radius = 285.88, P-index = 0.260. (Modified from Rasi et al, 2003.)... 

Experiments have been set up in this direction (Cheng and Luisi, 2003) utilizing extruded oleate vesicles having radii of 31.6 and 64.1 nm. For the same surfactant concentration, the total surface areas are the same for the two families of vesicles (neglecting the differences arising in aggregation number due to the differences... 

Oleic acid/oleate vesicles containing the enzyme Q(3 replicase, the RNA template and the ribonucleotides. The water-insoluble oleic anhydride was added externally. 

Rasi, S., Mavelli, F., and Luisi, P. L. (2003). Cooperative micelle binding and matrix effect in oleate vesicle formation. J. Phys. Chem. B, 107, 14068-76. 

Experiments have confirmed the idea that micelles as well as vesicles could grow autocatalytically (see [41] for a good overview). In a landmark paper Bachmann et al. [42] observed the formation of autocatalytically replicating micelles from sodium caprylate. The micelles could be converted into more stable vesiscles by pH change. Oleic acid/oleate vesicles can also mul-... 

The same kind of process can occur with vesicles. We have described this with oleate vesicles and other carboxylates, including giant vesicles. The case of vesicles is analytically more difficult, owing to the fact that upon hydrolysis of the water-insoluble ester or anhydride on the bilayer, two processes occur simultaneously the growth in size and the increase in population number. Both effects contribute to the increase of turbidity of the vesicle suspension, which is the most convenient way to measure the change of concentration of vesicles. As part of the... 

This effect has been studied in our group by Kenichi Morigaki in his dissertation. He utilized the hydrophobic tripeptide Z-Phe-His-Leu-NHj (Z = carboben-zyloxy). This compound has been shown before in the literature to display catalytic properties towards the hydrolysis of certain esters. The authors were particularly interested in the stereoselectivity of the process of L- towards D-amino acids and less in the enhancement of catalysis operated by the micelles. The substrate chosen was a very lipophylic ester, nitrophenylpalmitate. Morigaki used oleate vesicles, and later POPC liposomes, obtaining qualitatively similar results. ... 

PNPase has been entrapped inside extruded fatty acid vesicles, which are fed by externally added ADP and oleic anhydride. ADP is polymerized inside the vesicles, producing poly(A), whereas - simultaneously - oleate vesicles self-reproduce at the expenses of oleic anhydride. 

QP replicase has been entrapped within extruded oleate vesicles, together with a template RNA and NTPs. Vesicles are fed by externally added oleic anhydride, so that self-reproduction occurs. Simultaneously, RNA is replicated in the vesicle core. This is an example of core-and-shell reproduction. 

Figure 17.9 RNA replication inside self-reproducing oleate vesicles. Qfi replicase, an RNA-dependent RNA polymerase is entrapped inside oleate vesicles, in the presence of RNA template and NTPs. Copies of the RNA template are produced, and, simultaneously, oleate vesicles grow and divide according to the mechanism shown in Figure 17.6. In this way, internal RNA is replicated within self-reproducing vesicles. The mechanism stops when a daughter vesicles, produced by division of a previously grown vesicle, does not contain any QP replicase molecule. This vesicle dies due to the dilution of the catalyst. Notice that if solutes are always retained in every division step (i.e., without any loss in the environment) when only one QP replicase is present in a certain grown vesicle, the division will produce one functional daughter vesicle...

K. Ruiz-Mrrazo, P. Stano, P. L. Luisi, Lysozyme effect on oleic acid/oleate vesicles, J. Lipos. Res., 2006, 16, 143-154. 

P. Stano, E. WehrU, P. L. Luisi, Insights on the oleate vesicles self-reproduction, J. Phys. Condens. Matter, 2006, 18, S2231AS2238. 

Figure 3.11. DMPC/oleate vesicle diameter data at 15 and 35°C, as determined by photon correlation spectroscopy, showing results obtained from all concentrations measured [DMPCJfinai = 0.25 mmol dm- pH 8.5...

In contrast with phospholipid vesicles, the oleic acid-oleate vesicle system is characterized by a relatively high monomer solubility. The critical sodium oleate concentration for micelle formation (cmc) at about pH 10.5 is in the range 0.7-1.4mM [10] it has been reported that the monomer solubility at pH 7.4 is around 10-20 pM [11]. (For comparison, the monomer concentration of 1,2-dipalmitoyl-5n-glycero-3-phosphocholine (DPPC) is known to be aroimd 10 M [12].) Due to the relatively high monomer concentration, several physicochemical properties of oleic acid-oleate vesicles are different from vesicles made from phospholipids, such as phosphatidylcholines. The kinetics of lipid exchange is expected to be faster in the... 

In the present study, electron spin resonance (ESR) spectra of a fatty-acid spin probe (16-doxylstearic acid, see Figure 19.1) incorporated into an oleic acid-oleate system were measured in order to get insight into the pH-dependent aggregation properties of sodium oleate and oleic acid. Furthermore, the dilution-induced transformation of submicrometer-sized particles (micelles and/or vesicles) into giant oleic acid-oleate vesicles was investigated by electrophoretic light scattering measurements. 

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