It is well known that sterols can change the fluidity of the membrane, helping the cell to adapt to the surrounding environment making it vital for cell functions. Prokaryote cells lack sterols in the cell membrane, but research have found that hopanoids play a similar role. Tetrahydroxybacteriohopaneglycolipid (hopanoid) as well as betulinic acid and cholesterol were investigated to see whether there were any changes in the fluidity of the membrane, once these compounds were introduced. The measurements were taken using steady-state fluorescence polarisation. Betulinic acid and tetrahydroxybacteriohopaneglycolipid have similar structure to cholesterol, and both are thought to reduce the fluidity of the membrane. Fluorescent probe 1,6-Diphenyl-1,3,5-hexatriene was used to measure the movement of the membrane lipids. A Perkin-Elmer LS5B Luminescence Spectrometer with a polarisation attachment was used to excite the fluorescent probe, and then the readings were recorded. Betulinic acid was used as a positive control for comparison, were as the concentration of betulinic acid increased, the membrane became more stabilised. With the addition of cholesterol, the ordering effect was enhanced with betulinic acid. Tetrahydroxybacteriohopaneglycolipid also showed an ordering effect of the cell membrane. But comparing to cholesterol alone, tetrahydroxybacteriohopaneglycolipid didn't stabilise the membrane as much.
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1.1 Membrane Fluidity
Biological membranes are highly important in the control and maintenance of membrane fluidity. They are highly important for cell functions and cell integrity. They form heterogeneous, two-dimensional assemblies of lipids and proteins in the membranes. This allows the membranes to undergo processes such as cell signalling, where the cells can communicate with its environment; also that cell can adapt and make appropriate responses, if the surrounding environment changes (Halling et al., 2008). The cell membrane must first undergo lipid sorting, where the hydrophobic tail (hates water) and hydrophilic head (likes water) of the lipid molecules are in line together, forming a bi-layer structure (fig. 1.1.1)(Halling et al., 2008). It is well understood that eukaryotes cells contain cholesterol, that functions as a major modulator of lipid lateral segregation and packing density, making it a major factor for the permeability, and the fluidity of cell membranes (Mas-Oliva and Delgado-Coello, 2007). There is a very strong link between the fluidity of a cell membrane and the ratio of phospholipids to cholesterol. It been stated that the higher the concentration of cholesterol within the cell membrane the less movement or fluid the membrane becomes (Whiting et al., 1998). The structure of cholesterol shows a small polar head group, which allows the molecule to come close contact with phospholipids. This polar head group is able to form a hydrogen bond to the adjacent polar phospholipid heads. But because cholesterol is flat and has a rigid ring structure, it is able to align itself adjacent to the phospholipid tails thus able to influence the packing of surrounding lipids (fig 1.1.2) (Mas-Oliva and Delgado-Coello, 2007). Prokaryotes lack or have no sterols in their cell membranes, and there is an ongoing research into finding molecules that play a similar role to cholesterol in eukaryote cells. Pentacyclic triterpenoids such as hopanoids have been found to have a similar structure to cholesterol. They have been found in a number of aerobic bacteria such as heterotrophs and cyanobacteria, but they also been found in some anaerobic bacteria (Kannenberg and Poralla, 1999). These pentacyclic triterpenoids seem to carry out the same functions as cholesterol does in the cell membrane in eukaryote cells. They are shown to be sterol analogues, and in theory as the concentration of the hopanoids increases, the less fluidity the membrane becomes. If this is the case they might provide resistance to ethanol and heat shock stress, which cholesterol seem to provide in eukaryote cells (Welander et al., 2009).
Tetrahydroxybacteriohopaneglycolipid is a hopanoid that has a similar structure to cholesterol (Rohmer 1993). These hopanoids have been found in a wide range of prokaryote cells; mostly found in aerobic bacteria (Kannenberg and Poralla, 1999). Within these micro-orgamisms they have shown to play a role of membrane stabilisers, similar to the role played by cholesterol in eukaryote cells. It has been found that sterols such as cholesterol are rare in bacteria cells (Rohmer et al., 1979).
The elongated hopanoids such as tetrahydroxybacteriohopaneglycolipid have a very similar function to sterols (Rohmer 1993). They both condense phospholipids above the transition temperature (Rohmer et al., 1979) and presumably in biological membranes (Rohmer et al., 1979). Hopanoids are derived from the cyclisation of squalene (Ochs et al., 1990). If compared this to sterols, sterols are synthesised from squalene epoxide (Ochs et al., 1990). The synthesis of sterols is oxygen dependent, and within the membrane, a demethylation reaction occurs (Kannenberg and Poralla, 1999). This has lead to the theory, that perhaps sterol biosynthesis is a modern metabolic evolutionary process, compared to the primitive hopanoid biosynthesis (Flesch and Rohmer, 1988). For synthesis of hopanoids, squalene is cyclised into hopane derivative, which then leads to an amphiphilic product, diplopterol. This reaction requires H2O for the addition of a hydroxyl group, under anaerobic conditions (Flesch and Rohmer, 1988). This points further in to the theory that these reactions are primitive when comparing to the synthesis of cholesterol. Assuming that the atmosphere was oxygen-free, the OH group of the products could have derived from water, where in synthesis of cholesterol, it would have come from the air (Ourisson et al., 1979).
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Hopanoids have been reported to be located in higher plants, such as few mosses, and fungi (Rohmer et al., 1979; Poralla and Kannenberg, 1987). But plant hopanoids has shown to carry out no specific functions, so they are therefore classed as secondary metabolites (Blumenberg et al., 2006). Also there is no evidence that plants have squalene as their biosynthetic precursor. This points to the fact that perhaps different derivatives of hopanoids can form (Rohmer et al., 1979; Poralla and Kannenberg, 1987).
Hopanoids possess a quasi-planar, rigid, amphiphilic structure. This in itself has similar molecular dimensions to cholesterol (Kannenberg and Poralla, 1999). This amphiphilic structure increases the stability of the membrane by inserting into the phospholipids reducing fluidity and the permeability (Rohmer et al., 1979). What differs compared to cholesterol, is that the cyclo-hexane rings in hopanoids have chair-chair-chair-chair-chair conformations and in cholesterol it is a chair-boat-chair-boat-open conformations (Flesch and Rohmer, 1988)
Experiments have been done using a eukaryote cell protozoon Tetrahymena pyriformis. Tetrahymena pyriformis was grown in a medium containing sterols, which they concluded to beneficial for the cell. But once removed from the medium they found that Tetrahymena biosynthesised diplopterol, a hopanoid and also tetrahymanol a hopanoid like isomer. They found that tetrahymanol was localised in the membranes, adjacent to the phospholipid composition allowing proper fluidity (Rohmer et al., 1979). This lead to the possible conclusion that perhaps hopanoids play a sterol-like role in membranes of Tetrahymena, and it suggests clearly, that this may also be true in the prokaryotes that contain hopanoids (Rohmer et al., 1979). But they also pointed out that not all prokaryotes contain sterols or hopanoids in their membranes.
The property of stabilising the membrane could give hopanoids cytotoxic nature. This could lead to drugs that could be used to cause the cell to induce apoptosis, or programmed cell death. The cytotoxic properties could be used against cancer cells, without affecting other normal cells. This will result in the changes of the fluidity and the permeability of the cell membranes disrupting the transport of essential molecules through the cell membranes (Nagumo et al., 1991; Chen et al., 1995). There are more common cytotoxic drugs such as doxorubicin. This drug is used for cancer chemotherapy, were it acts by activating different signalling pathways, that results in apoptosis of the cell (Kroemer, 2001).
The study of hopanoids has pointed to the fact that there are three series of 3-deoxyhopane derivatives that have been found in living prokaryote cells (fig. 1.2.1). The simplest are derivatives of hopane, diploptene (hop-22(29)-ene) and diplopterol (hopan-22-ol), which are C30. A second family are composed of C35; derivatives of bacteriohopane (tetrahydroxybacteriohopane) (Rohmer et al., 1979). These derivatives of C35 are abundant throughout all hopanoid-producing bacteria that have been analysed so far (Blumenberg et al., 2006). Compare to the simplest forms, the C35 derivatives of bacteriohopane have an additional C5 unit linked by a carbon/carbon bound to the isopropyl group of the hopane framework (Rohmer 1993). This allows other groups to form replacing the hydroxyl group, such as aminotrihydroxybacteriophan which has amine group. Also aetrahydroxybacteriohopanglycolipid, which has a glycolipid group replacing the hydroxyl group on carbon 35 (fig 1.2.2).
1.3. Betulinic Acid
Betulinic acid is a compound that has been found in many plant species such as in the bark of the white birch tree (Betula alba). It contains five rings with a total of thirty carbon atoms, forming a pentacyclic triterpene molecule (fig 1.3.1.) Betulinic acid has cytotoxic properties, promoting apoptosis, or programmed cell death (Fulda et al., 1997). This could be applied to cancer cells without affecting other normal cells in the process (Fulda et al., 1997). It is thought to believe that betulinic acid promotes apoptosis by overproducing reactive oxygen species, affecting the mitochondria directly (Neuzil et al., 2007). Recent studies were put forward looking at, how betulinic acid affects the mitochondria which could induce apoptosis. Kroemer & Reed, (2000) suggested that, because the mitochondria is vital for the whole cell in providing energy to allow essential molecules in and out of the cell, but also preventing unwanted molecules from entering; affecting the permeability of the mitochondrial membranes internal proteins, such as cytochrome c and caspases , which could start the apoptosis pathway. Later in 2000 studies showed that betulinic acid induced apoptosis in neuroectodermal tumour cells (Fulda and Debatin, 2000).
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Their studies showed that betulinic acid reduced mitochondrial membrane potential, which then lead to apoptosis. They also found that the reduction of mitochondrial membrane potentially lead to the release of proteins, apoptosis-inducing factor (AIF) and cytochrome c in to the cytosol. This release of the two proteins will result in the cleavage of the effector caspase-3. Caspase-3 will then cleave Poly-ADP-ribose polymerase (PARP). Poly-ADP-ribose polymerase (PARP) is a protein that is involved in DNA repair. This cleavage of Poly-ADP-ribose polymerase (PARP) will eventually lead to cell death. Fulda and Debatin (2000) lead to the conclusion that targeting the mitochondria directly will kill the cell and could be incorporated into cancer cells
1.4. Aims and Objectives
Aim of the experiments that I will be conducting, will be to see if there is any change in membrane fluidity once tetrahydroxybacteriohopaneglycolipid (Hopanoid) have been introduced. The method will use steady-state fluorescence polarisation. This will measure any change in anisotropy, indicating any ordering effect in the membrane. A fluorescent probe will locate in-between the membrane bilayer then fluoresces indicating if there is a ordering effect or not. An artificial liposome will be used, from phospholipid phosphatidylcholine (PC); this would be extracted from egg yolk. A buffer will be added to allow the lipids to form in an aqueous environment with pH 7. This will allow the lipids to position themselves so that the hydrophilic head group and the hydrophobic hydrocarbon chain can a-line forming a bilayer structureÂ (Gurr et al, 2002,Halling et al., 2008). Tetrahydroxybacteriohopaneglycolipid will be added to the liposomes and the affects will be measured.Â Cholesterol will also be incorporated into the liposomes. This is to compare readings from cholesterol and tetrahydroxybacteriohopaneglycolipid. Also to see if there is any effect cholesterol has on betulinic acid. Hopanoids function is to order the membrane fluidity by monitoring the membrane permeability. Last year a study found that diplopterol a hopanoid, had no effect on membrane fluidity, even when the sample was purified. Diplopterol is found in bacteria cell membranes but is not the most abundant hopanoid. Tetrahydroxybacteriohopane (bacteriohopanetetrol) and its derivatives can be found in many aerobic and anaerobic bacteria and are the most abundant hopanoids. The introduction of Tetrahydroxybacteriohopaneglycolipid to the liposome membrane in theory will order the permeability by decreasing the membrane fluidity. There is very little work that has been done to support the theory. Figure 1.3.2 is a picture that is in theory suppose to happen once the addition of hopanoids into a membrane bilayer (Rohmer et al., 1979). So these experiments will answer the following questions: By introducing tetrahydroxybacteriohopaneglycolipid to the liposome, will this reduce the membrane fluidity? How does this compare to cholesterol? Will the liposome be affected if betulinic acid was added to an already ordered cell membrane?
3.1. Liposome Preparations
Figure 3.1.1 shows the results from the liposome preparations. Results were taken using different concentrations liposome suspension ranging from 0.05mg/ml to 0.500mg/ml. This experiment was carried out to the ideal concentration to use in the following experiments. The graph shows that, from 0.05mg/ml to 0.100mg/ml there is a decrease in anisotropy, as the lipid concentration increased in the cuvette. Then there is a plateau region from 0.100mg/ml to 0.200mg/ml. From 0.200mg/ml to 0.500mg/ml, there is a large drop where anisotropy value decreased by 0.03. From the graph the ideal concentration would be where the anisotropy values are constant, in the region of the plateau. For the rest of the experiments the concentration of the liposome suspension used was 0.2mg/ml.
3.2. Betulinic Acid Results
Figure 3.2.1 shows whether the solvent dimethyl sulphoxide (DMSO) can affect the fluidity of the membrane. Betulinic acid would e dissolved in DMSO, so experiments needed to be conducted, if there is any affect once DMSO is added to the liposome suspension. If there is change in anisotropy this would give conflicting results when measuring betulinic acid.
Volumes of DMSO ranged from 0Âµl to 100Âµl in the liposome suspension. From looking at the graph, there is a slight drop in anisotropy as the volumes of DMSO increased. The graph also shows that the volume range from 0Âµl to 50Âµl, did not show as much of an effect compared to the volumes range from 50Âµl to 100Âµl. The anisotropy value ranging from 0Âµl to 50Âµl only differed by 0.007r and the anisotropy value ranging 50Âµl to 100Âµl differed by 0.01r. So there is a decrease ranging from 0Âµl to 50Âµl, but the range from 50Âµl to 100Âµl is greater .From the graph, it is possible to come to the conclusion that no more than 50Î¼l of DMSO should be injected into the liposomes otherwise it will give false results when using betulinic acid.
Figure 3.2.3 shows the effect of betulinic acid on membrane fluidity. The graph shows that there was a steady rise in anisotropy from 0.106 to 0.160 as the concentration of betulinic acid increased. The concentration ranges from 0.00mM to 0.25mM. The data indicates that betulinic acid decreases membrane fluidity. Also the membrane becomes more ordered as the concentration of betulinic acid increases.
Figure 3.2.4 shows the change in the effect of betulinic acid on membrane fluidity with the addition of cholesterol into the liposome suspension. This experiment was conducted to see whether there would be greater ordering affect then betulinic acid alone. The cholesterol ratio stayed constant at 5:1
3.3. Tetrahydroxybacteriohopaneglycolipid results
Figure 3.3.1 shows the results of thin layer chromatography. TLC was carried out on the Tetrahydroxybacteriohopaneglycolipid solution. From looking at the TLC plate one spot can be seen. After calculating the Rf values using the equation:
Rf = Distance travelled by spot (mm)
Distance travelled by solvent (mm)
Tetrahydroxybacteriohopaneglycolipid was discovered with an of Rf value=0.26
Figure 3.3.2 shows the results obtained from membrane fluidity tests with methanol. Tetrahydroxybacteriohopaneglycolipid is able to dissolve in chloroform, dichloromethane, diethylether, and methanol. Chloroform, dichloromethane, diethylether are nonpolar compounds; so they are immiscible with water. This will affect the formation of the liposome, giving false readings. Methanol on the other hand is polar, making the compound miscible in water. The graph shows that methanol can cause a change in membrane fluidity. From the results obtained, there is a decrease in anisotropy as you increase the volume of methanol. There is a steady decrease in anisotropy from 0Î¼l to 15Î¼l, where the anisotropy difference is 0.003r. There overall drop in anisotropy was 0.008r. This is similar to the DMSO at volumes at 0Î¼l to 50Î¼l. The results with methanol were overlooked. Tetrahydroxybacteriohopaneglycolipid was then dissolved in methanol.
Figure 3.3.3 shows the effects of tetrahydroxybacteriohopaneglycolipid on membrane fluidity from a 0.5mM solution. There is very large drop in anisotropy as the concentration of Tetrahydroxybacteriohopaneglycolipid increased in the liposome suspension increased. The anisotropy value dropped from 0.121 to 0.106. This suggests that tetrahydroxybacteriohopaneglycolipid has affected the membrane becoming more fluid. The Concentration ranges from 0mM to 0.0125mM, which is very low, and there might not be any sufficient amount of the hopanoids to cause an effect on the membrane. This increase in fluidity may be due to the methanol.
Figure 3.3.4 shows the affects of tetrahydroxybacteriohopaneglycolipid on membrane fluidity. The concentration of the stock solution was increased to 1.8mM. The graph indicates that there is little change in anisotropy as the concentration of tetrahydroxybacteriohopaneglycolipid in the liposome suspension increased. The largest deviation in anisotropy is only 0.001r. There is little change in anisotropy as you increase the concentration of Tetrahydroxybacteriohopaneglycolipid in the liposome suspension. This suggests that at 1.8mM Tetrahydroxybacteriohopaneglycolipid has no effect on membrane fluidity. The largest concentration was 0.045mM
Figure 3.3.5 shows the effects of Tetrahydroxybacteriohopaneglycolipid on membrane fluidity. The concentration of the solution was increased to 4.6mM. There is slight increase in anisotropy value, as the concentration of Tetrahydroxybacteriohopaneglycolipid increases. The maximum concentration of the hopanoid is 0.115mM. There is an ordering effect but not enough to say that this hopanoid affects the cell membrane fluidity.
Figure 3.3.6 shows the change in the effect of Tetrahydroxybacteriohopaneglycolipid on membrane fluidity. This was an alternative method showing the anisotropy values. Instead of increasing the concentration of the hopanoid in the liposome suspension, instead it focuses on the ratio of the hopanoid: lipid molecules.
10mM solution was made and 5Âµl was added to the 3.8 Âµl phospholipid so that the ratio of Tetrahydroxybacteriohopaneglycolipid: lipid molecules was 10:1. Compare to liposome suspension with no hopanoids you can see that there is an ordering effect where the anisotropy increased from 0.114r to 0.135r, so there is a difference of 0.021r. This is a 19% increase in anisotropy value.
Figure 3.3.7 shows the change in the effect of Tetrahydroxybacteriohopaneglycolipid on membrane fluidity. 10mM solution was made and 5Âµl was added to the 1.9 Âµl phospholipid so that the ratio of Tetrahydroxybacteriohopaneglycolipid: lipid molecules was 5:1. If you compared the result to the liposome suspension with no hopanoids, there is an ordering effect were the anisotropy increased from 0.118r to 0.151r, so there is a difference of 0.033r. This is a 28% increase in anisotropy value.
The main aim of this project was to use the method of steady-state fluorescence polarisation, to see whether triterpenoids such as betulinic acid and tetrahydroxybacteriohopaneglycolipid (Hopanoid) will change the membrane fluidity. The project showed that the structure of the two triterpenoids that was tested played a significant role in the ordering of the membranes. Tests were done on cholesterol, to see whether it had a more ordering effect on the membrane then the hopanoid. Cholesterol was also added to the membrane to see whether, it causes further enhancement once betulinic acid was added. Results have already been recorded, showing that betulinic acid orders the membrane; this was used as a positive control for comparison. Due to the similarity of betulinic acid and cholesterol in their structure, tetrahydroxybacteriohopaneglycolipid was thought to order the membrane in the same way. The theory has stated that cholesterols in the membrane main function is to control membrane permeability and fluidity (Mas-Oliva and Delgado-Coello, 2007), and because tetrahydroxybacteriohopaneglycolipid has a similar structure to cholesterol, it might have the same properties.
It has been stated that tetrahydroxybacteriohopaneglycolipid (Hopanoid), will perform in the same way, based on the studies that have been done on hopanoids. Kannenberg and Poralla, (1999) found that hopanoids carry out the function in prokaryotes that sterols perform in eukaryotes cells. From looking at results, once tetrahydroxybacteriohopaneglycolipid was added to the membrane there was an ordering effect. There have been some studies that have been done, to find the effects of hopanoid on cell membrane fluidity. Last year research was carried out measuring the effect of diplopterol (Hopanoid), on cell membrane fluidity. The results were suppressing as it showed no positive or negative correlation, indicating that there is no effect on the membrane fluidity; despite the fact the sample was purified (fig 4.0.1).
Diplopterol is a hopanoid that is found in prokaryotes cell, but it is not the most abundant hopanoid. The elongated hopanoids such as bacteriohopane and its derivatives could itself order the membrane (Kannenberg and Poralla, 1999).
Diplopterol is similar to cholesterol in the structure, but comparing to the elongated hopanoids, the elongated hopanoids are very similar. Cholesterol has hydrocarbon tail that is bonded to a five carbon ring. This allows the molecule to partition between the phospholipid tails so that the membrane would not be able to move (Halling et al., 2008). But diplopterol does not have hydrocarbon chain and perhaps that is the reason why there was no change in fluidity (fig 4.0.2).
To measure the membrane fluidity, steady-state fluorescence polarisation was used. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was the fluorescent probe that was used to identify any change in the membrane fluidity. The probe partitions into the membrane bilayer in-between the hydrocarbon tails and once exited from the beam of polarised light, it emits fluorescence which is detected (fig 4.0.3). This technique is useful in identifying any changes in the membrane fluidity giving an anisotropy value. If there is strong signal in the emitted fluorescence, this states that the movement of the probe is restricted thus indicating a reduction in membrane fluidity. If there is an increase in the emitted fluorescence this indicates there is a reduction in the mobility of the probe therefore a decrease in the fluidity of the membrane. So as anisotropy increases, the membrane fluidity decreases and if anisotropy decreases the membrane fluidity increases (Shinitzky and Barenholz, 1978).
4.1. Preparation of the Liposomes
In order to use the right lipid concentration for the liposome suspension in all the experiments, tests were done to show, if the anisotropy value is affected by increasing the lipid concentration (fig 3.1.1). Figure 3.1.1 is an experiment that shows the lipid concentration against and the anisotropy values. There is a decrease from 0.050mg/ml to 0.100mg/ml in anisotropy, and then it seems to have a plateau region from 0.100mg/ml to 0.200mg/ml were the anisotropy value stayed constant. From 0.200mg/ml to 0.500mg/ml the anisotropy value then decreased once again. Theoretically as the lipid concentration increases this should not affect anisotropy. The decrease from 0.050mg/ml to 0.100mg/ml could be due to scattering, otherwise known as Raman Scattering. It was C. V. Raman that found, light is scattered from a molecule, and the photon that is scattered has the same energy as the light emitted. At low concentration of the lipids, the scattering is caused by the aqueous environment in the cuvette. This causes interference in the frequency and wavelength, showing a high anisotropy value (Banwell & McCash, 1994). From 0.200mg/ml to 0.500mg/ml the anisotropy value decreased. This is caused by light scattering by the increase in lipid concentration. The concentration of the lipid in the cuvette is high and the light emitted is scattered at different angles instead of the light passing through the solution to the detector (Banwell & McCash, 1994). At 0.100mg/ml to 0.200mg/ml the anisotropy values are constant, showing plateau region. It is within this region there is no Raman Scattering, and the light emitted is not scattered by the molecules in the cuvette (Banwell & McCash, 1994). As there is no interface from the outside environment this result is reliable and the concentration of the lipid for all the experiments that were conducted was at 0.200mg/ml.
4.2. Betulinic Acid Measurements
The experiments with betulinic acid were used as a comparative control. DMSO was the solvent for which betulinic acid was dissolved in. Before tests that could be done, to see if betulinic acid ordered the membrane, experiments needed to be conducted to see whether DMSO affected the membrane alone. Figure 3.2.1, shows a drop in anisotropy value as the volume of DMSO increased. There was drop from 50Âµl to 100Âµl by 0.014, indicating that the membrane becoming more fluid. But this decrease in anisotropy is small and will not affect the overall result once betulinic acid it added to the membrane.
Betulinic acid partitions in the cell membrane, because in the aqueous solution, it is thermodynamically unfavourable. The structure is similar to cholesterol, which could explain figure 3.2.3, showing an increase in anisotropy value by 58%. As the concentration of betulinic acid increases the membrane becomes more ordered. Looking at the structure of betulinic acid (fig.1.2.1.) there are two polar end groups at opposite ends; the polar hydroxyl (OH) group and a carboxyl (COOH) group. The carboxyl group are more polar then the hydroxyl group, and is likely to insert itself to the head group of the phospholipid and the hydroxyl group will lie along side with the phospholipid tails. This could explain why betulinic acid as the same properties as cholesterol in ordering the membrane.
Experiments with cholesterol and betulinic acid reduced the membrane fluidity more effectively then betulinic acid alone.
Cholesterol was added to the liposome suspension, at a ratio of 5:1, which was constant throughout the whole experiment. Figure 3.2.4 shows that there is a higher decrease in membrane fluidity once betulinic acid is added. Comparing to figure 3.2.3, where it was just betulinic acid that was added to the liposome suspension, the cholesterol and betulinic acid showed an increase in ordering of the membrane, but not by much. There was only a 28% increase in anisotropy in figure 3.2.4 as the concentration of betulinic acid increases; comparing to the betulinic acid alone it was a lot higher at 58%.
Figure 3.2.4 shows the addition of betulinic acid to a membrane that is already contains cholesterol at a 5:1 ratio. It is well understood that cholesterol stabilises membrane, reducing cell membrane fluidity (Ikonen, 2008) and at a ratio of 5:1, the membrane is tightly packed and ready ordered, making it difficult for betulinic acid to partition in to the membrane. The addition of betulinic acid therefore did not make much of an impact on membrane fluidity has thought.
It would be interesting to see if betulinic acid was added first at a 5:1 ratio, then cholesterol at different concentrations. But this causes problems as cholesterol dissolves in chloroform and methanol at a 2:1 ratio. Chloroform is a nonpolar compound; thus making it immiscible with water. If chloroform was added to the liposome suspension, this will disrupt the formation of the liposome and give false results.
4.3. Tetrahydroxybacteriohopaneglycolipid Measurements
Thin layer chromatography was preformed to see whether tetrahydroxybacteriohopaneglycolipid contained any impurities. The results from figure 3.3.1 showed one spot on the TLC plate. The Rf value was calculated to be 0.26, using the equation in section 3. The Rf value for tetrahydroxybacteriohopaneglycolipid, that was suggested by Dr Günther Muth was to be 0.26, which relates to the spot that was on the TLC plate, leading to conclusion that the sample was pure.
For the experiments measuring the anisotropy; methanol was the solvent, were tetrahydroxybacteriohopaneglycolipid was dissolves in, as recommended by the hopanoid supplier (Dr Günther Muth, University of Tubingen). Methanol was then tested to see if it had any effect on the membrane fluidity. Figure 3.3.2 shows a drop in anisotropy as volumes of methanol increases. Methanol increases the membrane fluidity, but the affect was not large enough to cause any interface, so the experiments with tetrahydroxybacteriohopaneglycolipid carried on.
Figure 3.3.3 shows the affect tetrahydroxybacteriohopaneglycolipid has on the membrane fluidity. As the concentration of tetrahydroxybacteriohopaneglycolipid increases the anisotropy value drops from 0.121 to 0.106, decreasing by 15%. This was unexpected as it indicates that the membrane is becoming less ordered as the concentration of the hopanoid increases. The stock solution was 0.5mM making the maximum concentration from figure 3.3.3, 0.0125mM. This gives a ratio of hopanoid: lipid molecules of 100:1.
This was investigated further using another method, were the hopanoid was incorporated in to the PC, and then it was subjected to oxygen free nitrogen (OFN) before the buffer was added.
The alternative method will show any changes in anisotropy depending on the ratio of hopanoid: lipid molecules. The graph below shows a control with no hopanoids and also a comparative result with hopanoids, were a liposome suspension was made that contained a ratio of hopanoid: lipid molecules of 100:1, similar to the maximum concentration from figure 3.3.3. From looking at the graph below, there is no change in the anisotropy value.
This experiment was then performed again, but the ratio of hopanoid: a lipid molecule of 200:1.
The results above showed no difference in anisotropy value, meaning there is no change in the fluidity of the membrane. This makes sense because introducing a compound that has a similar structure to cholesterol should not increase the fluidity of the membrane; also that there literature did not support this finding. But figure 3.3.3 shows a decrease in anisotropy, this could be explained by methanol could have caused the membrane to become more fluid. At very low concentration of hopanoid and high volumes of methanol this could be the main reason why there was a decrease in figure 3.3.3.
Figure 3.3.3, 4.2.1 and 4.2.2 readings showed, at low concentrations tetrahydroxybacteriohopaneglycolipid had no effect on the membrane fluidity.
Another stock solution was made were the concentration was 1.8mM. Figure 3.3.4 shows there is no change on the membrane fluidity, as the concentration of tetrahydroxybacteriohopaneglycolipid increased. The highest concentration of tetrahydroxybacteriohopaneglycolipid in the cuvette is 0.045mM; this gives a ratio of hopanoid: lipid molecules of 30:1. This was unexpected because, at a ratio of cholesterol: lipid molecule of 30:1, there would be a slight ordering affect.
Figure 3.3.5 showed a positive correlation in anisotropy value as the concentration of tetrahydroxybacteriohopaneglycolipid increases. It increased by 7%, were the maximum concentration of tetrahydroxybacteriohopaneglycolipid is 0.115mM giving a ratio of hopanoid: lipid molecules of 15:1. This showed that at a concentration ranging from 0.045mM to 0.115mM causes the membrane to become more ordered.
This was investigated further using an alternative method, were Instead of measuring the anisotropy at different concentrations, the liposome suspension was made so that the hopanoid: a lipid molecule was at a ratio of 10:1, as described in section 2.5.
Figures 3.3.6 shows an increase of 18% in anisotropy. This provides evidence that tetrahydroxybacteriohopaneglycolipid orders the membrane if the ratio of hopanoid: lipid molecules is 10:1.
Another experiment was conducted to see whether there is further enhancement in ordering the membrane at the ratio of 5:1. Figure 3.3.7 showed an increase of 28% compared to the control. Figures 3.3.6 and 3.3.7 both showed that tetrahydroxybacteriohopaneglycolipid reduces the membrane fluidity. It also shows that at a ratio of 15:1 to 5:1 the membrane becomes more ordered compared to 100:1 to 30:1.
This can be explained by the structure of tetrahydroxybacteriohopaneglycolipid (fig. 1.2.1). The theory has stated that tetrahydroxybacteriohopaneglycolipid contains similar properties to cholesterol in ordering the membrane. Tetrahydroxybacteriohopaneglycolipid's structure is similar to cholesterol as both have a hydrocarbon chain and also they both contain a ring structure that stabilises the whole molecule. Tetrahydroxybacteriohopaneglycolipid contain an isopropyl chain were three hydroxyl (OH) groups are bonded covalently on carbon 32, 33 and 34. These polar head groups are likely to form hydrogen bonds with the phospholipid head groups and the surrounding aqueous environment. To be able to form the hydrogen bond, the hydroxyl (OH) group on the hopanoid must be in a Î² conformation, facing the C=O group on the head of the phospholipid. The cyclo-hexane rings at the end of the molecule will associate with the phospholipid tails, reducing their movement. This prevents the hydrocarbon chain to rotate 120Â° clockwise or anticlockwise forming a kink otherwise known as gauche conformation. Introducing an amphiphilic molecule such as cholesterol, betulinic acid and tetrahydroxybacteriohopaneglycolipid this reduces the ability of the phospholipid molecule to form a kink in the chain (Whiting et al., 2000).
An experiment was done using cholesterol with the same method above. Figure 4.2.3 shows the anisotropy value increased by 41%, at cholesterol: lipid molecule ratio of 5:1. Compared to the hopanoid 5:1 ratio, cholesterol stabilises the membrane more effectively then the hopanoid.
This can be explained, were most eukaryote cells are composed of phosphatidylcholine forming liposomes, most prokaryote membranes are mainly composed of phosphatidylethanolamine and phosphatidylglycerol. These phospholipids are able to form inverted micelles were hydrophilic heads points inwards (Gurr et al, 2002). This difference could explain why tetrahydroxybacteriohopaneglycolipid didn't show as much as an ordering effect as cholesterol did. It may be the difference in composition of the head group of the phospholipids, why tetrahydroxybacteriohopaneglycolipid didn't have the same effect. Another fact to point out is that tetrahydroxybacteriohopaneglycolipid contains a glycolipid which is hydrophilic at the end of the isopropyl chain. Perhaps it is this attachment, that maybe the reason why tetrahydroxybacteriohopaneglycolipid doesn't partition into the membrane bilayer as easily as cholesterol.
Studies have reported that tetrahydroxybacteriohopaneglycolipid has the same function as cholesterol in the cell membrane (Poralla, Kannenberg et al. 1980). They studied the bacteria Bacillus acidocaldarius, which was unique in that it grows at pH 3 and 65Â°C. They found that 15% of the lipids contain tetrahydroxybacteriohopaneglycolipid that might have contributed to the thermophilic and acidophilic properties in Bacillus acidocaldarius. This would make sense, because reducing the permeability of the membrane will reduce the passive diffusion of the protons and other solutes.
Betulinic acid has already been shown that it has cytotoxic properties. This in itself can induce apoptosis in cancer cells (Gopal et al., 2005). Future work can be done, to see how betulinic acid would be able to target cells, mainly cancer cells. A problem that is associated with betulinic acid is that it's very harmful to the body. It has the properties to induce apoptosis in cancer cells, but also other normal cells will be affected if not properly monitored. But before betulinic acid can be used as a drug, tests need to be done to show if the environment such as temperature and pH has an effect on betulinic acid (Immordino et al., 2006). Like hopanoids, betulinic acid has derivatives that have also been found in plants. It would be interesting to see if derivatives such as betulin and dihydrobetuline acid can affect the membrane.
There were three more hopanoids that was gained from Dr Günther Muth. It would be very interesting to see and compare hopanoids that have an isopropyl chain and hopanoids that don't on membrane fluidity. Hopene the simplest hopanoid, that could be compare to tetrahydroxybacteriohopane the simplest hopanoid with the isopropyl chain. This then will indicate if it is the hydroxyl group on the isopropyl chain and the chain itself, which causes the stabilisation property.
Hopanoids with different groups attached could also affect the cell membrane such as aminotrihydroxybacteriophan which contains a hydrocarbon chain, but also has a glucosamine group bonded. It would be interesting to see if other derivatives like aminotrihydroxybacteriophan will cause a more or less ordering effect to the membrane.
The property of stabilising the membrane could give the hopanoid a cytotoxic nature; changing the fluidity and the permeability of the cell membrane could disrupt the transport of essential molecules through the cell membranes.
The lipid fluidity can be measured in a number of ways. Some are costly but more sensitivity and some are cheap with less sensitivity. Nuclear magnetic resonance is a powerful experimental technique that can highlight any aspects of the lipid bilayer modification. The phospholipids will be treated with 31P or 2H and NMR spectroscopy gives the information on changes in the structure and dynamics of the phospholipids of the model lipid membrane. If there are any changes in the membrane fluidity, it will show peaks, and if the peaks are further apart, the membrane is less fluidised. Another method that can be used is colorimetric enzyme assay. This method does not measure the fluidity of the membrane, but measure the contents in the membrane. For example it can be used to see whether molecule partitions into the membrane, and then the fluidity can be measured using steady state fluorescence polarisation.