It is good known that steroid alcohols can alter the fluidness of the membrane, assisting the cell to accommodate to the environing environment doing it critical for cell maps. Prokaryote cells lack steroid alcohols in the cell membrane, but research have found that hopanoids play a similar function. Tetrahydroxybacteriohopaneglycolipid ( hopanoid ) every bit good as betulinic acid and cholesterin were investigated to see whether there were any alterations in the fluidness of the membrane, one time these compounds were introduced. The measurings were taken utilizing steady-state fluorescence polarization. Betulinic acid and tetrahydroxybacteriohopaneglycolipid have similar construction to cholesterol, and both are thought to cut down the fluidness of the membrane. Fluorescent investigation 1,6-Diphenyl-1,3,5-hexatriene was used to mensurate the motion of the membrane lipoids. A Perkin-Elmer LS5B Luminescence Spectrometer with a polarization fond regard was used to excite the fluorescent investigation, and so the readings were recorded. Betulinic acid was used as a positive control for comparing, were as the concentration of betulinic acid increased, the membrane became more stabilized. With the add-on of cholesterin, the telling consequence was enhanced with betulinic acid. Tetrahydroxybacteriohopaneglycolipid besides showed an telling consequence of the cell membrane. But comparing to cholesterol entirely, tetrahydroxybacteriohopaneglycolipid did n’t brace the membrane as much.
1.1 Membrane Fluidity
Biological membranes are extremely of import in the control and care of membrane fluidness. They are extremely of import for cell maps and cell unity. They form heterogenous, planar assemblies of lipoids and proteins in the membranes. This allows the membranes to undergo procedures such as cell signalling, where the cells can pass on with its environment ; besides that cell can accommodate and do appropriate responses, if the environing environment alterations ( Halling et al. , 2008 ) . The cell membrane must first undergo lipid sorting, where the hydrophobic tail ( hates H2O ) and hydrophilic caput ( likes H2O ) of the lipid molecules are in line together, organizing a bi-layer construction ( fig. 1.1.1 ) ( Halling et al. , 2008 ) . It is good understood that eukaryotes cells contain cholesterin, that functions as a major modulator of lipid sidelong segregation and packing denseness, doing it a major factor for the permeableness, and the fluidness of cell membranes ( Mas-Oliva and Delgado-Coello, 2007 ) . There is a really strong nexus between the fluidness of a cell membrane and the ratio of phospholipids to cholesterol. It been stated that the higher the concentration of cholesterin within the cell membrane the less motion or fluid the membrane becomes ( Whitening et al. , 1998 ) . The construction of cholesterin shows a little polar caput group, which allows the molecule to come close contact with phospholipids. This polar caput group is able to organize a H bond to the next polar phospholipid caputs. But because cholesterin is level and has a stiff ring construction, it is able to aline itself next to the phospholipid dress suits therefore able to act upon the wadding of environing lipoids ( fig 1.1.2 ) ( Mas-Oliva and Delgado-Coello, 2007 ) . Prokaryotes deficiency or have no steroid alcohols in their cell membranes, and there is an on-going research into happening molecules that play a similar function to cholesterol in eukaryote cells. Pentacyclic triterpenoids such as hopanoids have been found to hold a similar construction to cholesterol. They have been found in a figure of aerophilic bacteriums such as heterotrophs and blue-green algae, but they besides been found in some anaerobiotic bacteriums ( Kannenberg and Poralla, 1999 ) . These pentacyclic triterpenoids seem to transport out the same maps as cholesterin does in the cell membrane in eukaryote cells. They are shown to be sterol parallels, and in theory as the concentration of the hopanoids additions, the less fluidness the membrane becomes. If this is the instance they might supply opposition to ethanol and heat daze emphasis, which cholesterol seem to supply in eukaryote cells ( Welander et al. , 2009 ) .
Tetrahydroxybacteriohopaneglycolipid is a hopanoid that has a similar construction to cholesterol ( Rohmer 1993 ) . These hopanoids have been found in a broad scope of procaryote cells ; largely found in aerophilic bacteriums ( Kannenberg and Poralla, 1999 ) . Within these micro-orgamisms they have shown to play a function of membrane stabilizers, similar to the function played by cholesterin in eukaryote cells. It has been found that steroid alcohols such as cholesterin are rare in bacteriums cells ( Rohmer et al. , 1979 ) .
The extended hopanoids such as tetrahydroxybacteriohopaneglycolipid have a really similar map to sterols ( Rohmer 1993 ) . They both condense phospholipids above the passage temperature ( Rohmer et al. , 1979 ) and presumptively in biological membranes ( Rohmer et al. , 1979 ) . Hopanoids are derived from the cyclisation of squalene ( Ochs et al. , 1990 ) . If compared this to sterols, steroid alcohols are synthesised from squalene epoxide ( Ochs et al. , 1990 ) . The synthesis of steroid alcohols is oxygen dependant, and within the membrane, a demethylation reaction occurs ( Kannenberg and Poralla, 1999 ) . This has lead to the theory, that possibly sterol biogenesis is a modern metabolic evolutionary procedure, compared to the crude hopanoid biogenesis ( Flesch and Rohmer, 1988 ) . For synthesis of hopanoids, squalene is cyclised into hopane derived function, which so leads to an amphiphilic merchandise, diplopterol. This reaction requires H2O for the add-on of a hydroxyl group, under anaerobiotic conditions ( Flesch and Rohmer, 1988 ) . This points further in to the theory that these reactions are crude when comparing to the synthesis of cholesterin. Assuming that the ambiance was oxygen-free, the OH group of the merchandises could hold derived from H2O, where in synthesis of cholesterin, it would hold come from the air ( Ourisson et al. , 1979 ) .
Hopanoids have been reported to be located in higher workss, such as few mosses, and Fungis ( Rohmer et al. , 1979 ; Poralla and Kannenberg, 1987 ) . But works hopanoids has shown to transport out no specific maps, so they are hence classed as secondary metabolites ( Blumenberg et al. , 2006 ) . Besides there is no grounds that workss have squalene as their biosynthetic precursor. This points to the fact that possibly different derived functions of hopanoids can organize ( Rohmer et al. , 1979 ; Poralla and Kannenberg, 1987 ) .
Hopanoids possess a quasi-planar, stiff, amphiphilic construction. This in itself has similar molecular dimensions to cholesterol ( Kannenberg and Poralla, 1999 ) . This amphiphilic construction increases the stableness of the membrane by infixing into the phospholipids cut downing fluidness and the permeableness ( 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 cholesterin it is a chair-boat-chair-boat-open conformations ( Flesch and Rohmer, 1988 )
Experiments have been done utilizing a eucaryote cell protozoan Tetrahymena pyriformis. Tetrahymena pyriformis was grown in a medium containing steroid alcohols, which they concluded to beneficial for the cell. But one time removed from the medium they found that Tetrahymena biosynthesised diplopterol, a hopanoid and besides tetrahymanol a hopanoid like isomer. They found that tetrahymanol was localised in the membranes, next to the phospholipid composing leting proper fluidness ( Rohmer et al. , 1979 ) . This lead to the possible decision that possibly hopanoids play a sterol-like function in membranes of Tetrahymena, and it suggests clearly, that this may besides be true in the procaryotes that contain hopanoids ( Rohmer et al. , 1979 ) . But they besides pointed out that non all procaryotes contain steroid alcohols or hopanoids in their membranes.
The belongings of bracing the membrane could give hopanoids cytotoxic nature. This could take to drugs that could be used to do the cell to bring on programmed cell death, or programmed cell decease. The cytotoxic belongingss could be used against malignant neoplastic disease cells, without impacting other normal cells. This will ensue in the alterations of the fluidness and the permeableness of the cell membranes interrupting the conveyance of indispensable 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 malignant neoplastic disease chemotherapy, were it acts by triping different signalling tracts, that consequences in programmed cell death of the cell ( Kroemer, 2001 ) .
The survey of hopanoids has pointed to the fact that there are three series of 3-deoxyhopane derived functions that have been found in populating procaryote cells ( fig. 1.2.1 ) . The simplest are derived functions of hopane, diploptene ( hop-22 ( 29 ) -ene ) and diplopterol ( hopan-22-ol ) , which are C30. A 2nd household are composed of C35 ; derived functions of bacteriohopane ( tetrahydroxybacteriohopane ) ( Rohmer et al. , 1979 ) . These derived functions of C35 are abundant throughout all hopanoid-producing bacteriums that have been analysed so far ( Blumenberg et al. , 2006 ) . Compare to the simplest signifiers, the C35 derived functions of bacteriohopane have an extra C5 unit linked by a carbon/carbon edge to the isopropyl group of the hopane model ( Rohmer 1993 ) . This allows other groups to organize replacing the hydroxyl group, such as aminotrihydroxybacteriophan which has aminoalkane group. Besides aetrahydroxybacteriohopanglycolipid, which has a glycolipid group replacing the hydroxyl group on C 35 ( fig 1.2.2 ) .
1.3. Betulinic Acid
Betulinic acid is a compound that has been found in many works species such as in the bark of the white birch tree ( Betula alba ) . It contains five rings with a sum of 30 C atoms, organizing a pentacyclic triterpene molecule ( fig 1.3.1. ) Betulinic acid has cytotoxic belongingss, advancing programmed cell death, or programmed cell decease ( Fulda et al. , 1997 ) . This could be applied to malignant neoplastic disease cells without impacting other normal cells in the procedure ( Fulda et al. , 1997 ) . It is thought to believe that betulinic acid promotes programmed cell death by overproducing reactive O species, impacting the chondriosomes straight ( Neuzil et al. , 2007 ) . Recent surveies were put frontward looking at, how betulinic acid affects the chondriosome which could bring on programmed cell death. Kroemer & A ; Reed, ( 2000 ) suggested that, because the chondriosome is critical for the whole cell in supplying energy to let indispensable molecules in and out of the cell, but besides forestalling unwanted molecules from come ining ; impacting the permeableness of the mitochondrial membranes internal proteins, such as cytochrome degree Celsius and caspases, which could get down the programmed cell death tract. Subsequently in 2000 surveies showed that betulinic acid induced programmed cell death in neuroectodermal tumor cells ( Fulda and Debatin, 2000 ) .
Their surveies showed that betulinic acid reduced mitochondrial membrane potency, which so lead to apoptosis. They besides found that the decrease of mitochondrial membrane potentially lead to the release of proteins, apoptosis-inducing factor ( AIF ) and cytochrome degree Celsius in to the cytosol. This release of the two proteins will ensue in the cleavage of the effecter caspase-3. Caspase-3 will so split Poly-ADP-ribose polymerase ( PARP ) . Poly-ADP-ribose polymerase ( PARP ) is a protein that is involved in DNA fix. This cleavage of Poly-ADP-ribose polymerase ( PARP ) will finally take to cell decease. Fulda and Debatin ( 2000 ) lead to the decision that aiming the chondriosome straight will kill the cell and could be incorporated into malignant neoplastic disease cells
1.4. Purposes and Aims
Aim of the experiments that I will be carry oning, will be to see if there is any alteration in membrane fluidness one time tetrahydroxybacteriohopaneglycolipid ( Hopanoid ) have been introduced. The method will utilize steady-state fluorescence polarization. This will mensurate any alteration in anisotropy, bespeaking any telling consequence in the membrane. A fluorescent investigation will turn up mediate the membrane bilayer so fluoresces bespeaking if there is a telling consequence or non. An unreal liposome will be used, from phospholipid phosphatidylcholine ( Personal computer ) ; this would be extracted from egg yolk. A buffer will be added to let the lipoids to organize in an aqueous environment with pH 7. This will let the lipoids to place themselves so that the hydrophilic caput group and the hydrophobic hydrocarbon concatenation can a-line organizing a bilayer structureA ( Gurr et al, 2002, Halling et al. , 2008 ) . Tetrahydroxybacteriohopaneglycolipid will be added to the liposomes and the affects will be measured.A Cholesterol will besides be incorporated into the liposomes. This is to compare readings from cholesterin and tetrahydroxybacteriohopaneglycolipid. Besides to see if there is any consequence cholesterin has on betulinic acid. Hopanoids map is to order the membrane fluidness by supervising the membrane permeableness. Last twelvemonth a survey found that diplopterol a hopanoid, had no consequence on membrane fluidness, even when the sample was purified. Diplopterol is found in bacteriums cell membranes but is non the most abundant hopanoid. Tetrahydroxybacteriohopane ( bacteriohopanetetrol ) and its derived functions can be found in many aerophilic and anaerobiotic bacteriums and are the most abundant hopanoids. The debut of Tetrahydroxybacteriohopaneglycolipid to the liposome membrane in theory will order the permeableness by diminishing the membrane fluidness. There is really small work that has been done to back up the theory. Figure 1.3.2 is a image that is in theory suppose to go on one time the add-on of hopanoids into a membrane bilayer ( Rohmer et al. , 1979 ) . So these experiments will reply the undermentioned inquiries: By presenting tetrahydroxybacteriohopaneglycolipid to the liposome, will this cut down the membrane fluidness? How does this comparison 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 consequences from the liposome readyings. Consequences were taken utilizing different concentrations liposome suspension runing from 0.05mg/ml to 0.500mg/ml. This experiment was carried out to the ideal concentration to utilize in the undermentioned experiments. The graph shows that, from 0.05mg/ml to 0.100mg/ml there is a lessening in anisotropy, as the lipid concentration increased in the cuvette. Then there is a plateau part from 0.100mg/ml to 0.200mg/ml. From 0.200mg/ml to 0.500mg/ml, there is a big bead where anisotropy value decreased by 0.03. From the graph the ideal concentration would be where the anisotropy values are changeless, in the part of the tableland. For the remainder 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 impact the fluidness of the membrane. Betulinic acid would e dissolved in DMSO, so experiments needed to be conducted, if there is any affect one time DMSO is added to the liposome suspension. If there is alteration in anisotropy this would give conflicting consequences when mensurating betulinic acid.
Volumes of DMSO ranged from 0Aµl to 100Aµl in the liposome suspension. From looking at the graph, there is a little bead in anisotropy as the volumes of DMSO increased. The graph besides shows that the volume scope from 0Aµl to 50Aµl, did non demo as much of an consequence compared to the volumes range from 50Aµl to 100Aµl. The anisotropy value runing from 0Aµl to 50Aµl merely differed by 0.007r and the anisotropy value runing 50Aµl to 100Aµl differed by 0.01r. So there is a lessening runing from 0Aµl to 50Aµl, but the scope from 50Aµl to 100Aµl is greater.From the graph, it is possible to come to the decision that no more than 50I?l of DMSO should be injected into the liposomes otherwise it will give false consequences when utilizing betulinic acid.
Figure 3.2.3 shows the consequence of betulinic acid on membrane fluidness. 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 information indicates that betulinic acid decreases membrane fluidness. Besides the membrane becomes more ordered as the concentration of betulinic acid additions.
Figure 3.2.4 shows the alteration in the consequence of betulinic acid on membrane fluidness with the add-on of cholesterin into the liposome suspension. This experiment was conducted to see whether there would be greater telling affect so betulinic acid entirely. The cholesterin ratio stayed changeless at 5:1
3.3. Tetrahydroxybacteriohopaneglycolipid consequences
Figure 3.3.1 shows the consequences of thin bed chromatography. TLC was carried out on the Tetrahydroxybacteriohopaneglycolipid solution. From looking at the TLC home base one topographic point can be seen. After ciphering the Rf values utilizing the equation:
Rf = Distance travelled by topographic point ( millimeter )
Distance travelled by dissolver ( millimeter )
Tetrahydroxybacteriohopaneglycolipid was discovered with an of Rf value=0.26
Figure 3.3.2 shows the consequences obtained from membrane fluidness trials with methyl alcohol. Tetrahydroxybacteriohopaneglycolipid is able to fade out in trichloromethane, methylene chloride, diethylether, and methyl alcohol. Chloroform, methylene chloride, diethylether are nonionic compounds ; so they are non-miscible with H2O. This will impact the formation of the liposome, giving false readings. Methanol on the other manus is polar, doing the compound miscible in H2O. The graph shows that methyl alcohol can do a alteration in membrane fluidness. From the consequences obtained, there is a lessening in anisotropy as you increase the volume of methyl alcohol. There is a steady lessening in anisotropy from 0I?l to 15I?l, where the anisotropy difference is 0.003r. There overall bead in anisotropy was 0.008r. This is similar to the DMSO at volumes at 0I?l to 50I?l. The consequences with methyl alcohols were overlooked. Tetrahydroxybacteriohopaneglycolipid was so dissolved in methyl alcohol.
Figure 3.3.3 shows the effects of tetrahydroxybacteriohopaneglycolipid on membrane fluidness from a 0.5mM solution. There is really big bead 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 going more fluid. The Concentration ranges from 0mM to 0.0125mM, which is really low, and at that place might non be any sufficient sum of the hopanoids to do an consequence on the membrane. This addition in fluidness may be due to the methyl alcohol.
Figure 3.3.4 shows the affects of tetrahydroxybacteriohopaneglycolipid on membrane fluidness. The concentration of the stock solution was increased to 1.8mM. The graph indicates that there is small alteration in anisotropy as the concentration of tetrahydroxybacteriohopaneglycolipid in the liposome suspension increased. The largest divergence in anisotropy is merely 0.001r. There is small alteration in anisotropy as you increase the concentration of Tetrahydroxybacteriohopaneglycolipid in the liposome suspension. This suggests that at 1.8mM Tetrahydroxybacteriohopaneglycolipid has no consequence on membrane fluidness. The largest concentration was 0.045mM
Figure 3.3.5 shows the effects of Tetrahydroxybacteriohopaneglycolipid on membrane fluidness. The concentration of the solution was increased to 4.6mM. There is little addition in anisotropy value, as the concentration of Tetrahydroxybacteriohopaneglycolipid additions. The maximal concentration of the hopanoid is 0.115mM. There is an ordering consequence but non plenty to state that this hopanoid affects the cell membrane fluidness.
Figure 3.3.6 shows the alteration in the consequence of Tetrahydroxybacteriohopaneglycolipid on membrane fluidness. This was an alternate method demoing the anisotropy values. Alternatively of increasing the concentration of the hopanoid in the liposome suspension, alternatively it focuses on the ratio of the hopanoid: lipid molecules.
10mM solution was made and 5Aµl was added to the 3.8 Aµl 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 telling consequence where the anisotropy increased from 0.114r to 0.135r, so there is a difference of 0.021r. This is a 19 % addition in anisotropy value.
Figure 3.3.7 shows the alteration in the consequence of Tetrahydroxybacteriohopaneglycolipid on membrane fluidness. 10mM solution was made and 5Aµl was added to the 1.9 Aµl phospholipid so that the ratio of Tetrahydroxybacteriohopaneglycolipid: lipid molecules was 5:1. If you compared the consequence to the liposome suspension with no hopanoids, there is an telling consequence were the anisotropy increased from 0.118r to 0.151r, so there is a difference of 0.033r. This is a 28 % addition in anisotropy value.
The chief purpose of this undertaking was to utilize the method of steady-state fluorescence polarization, to see whether triterpenoids such as betulinic acid and tetrahydroxybacteriohopaneglycolipid ( Hopanoid ) will alter the membrane fluidness. The undertaking showed that the construction of the two triterpenoids that was tested played a important function in the ordination of the membranes. Trials were done on cholesterin, to see whether it had a more ordering consequence on the membrane so the hopanoid. Cholesterol was besides added to the membrane to see whether, it causes farther sweetening one time betulinic acid was added. Consequences have already been recorded, demoing that betulinic acid orders the membrane ; this was used as a positive control for comparing. Due to the similarity of betulinic acid and cholesterin in their construction, tetrahydroxybacteriohopaneglycolipid was thought to order the membrane in the same manner. The theory has stated that cholesterins in the membrane chief map is to command membrane permeableness and fluidness ( Mas-Oliva and Delgado-Coello, 2007 ) , and because tetrahydroxybacteriohopaneglycolipid has a similar construction to cholesterol, it might hold the same belongingss.
It has been stated that tetrahydroxybacteriohopaneglycolipid ( Hopanoid ) , will execute in the same manner, based on the surveies that have been done on hopanoids. Kannenberg and Poralla, ( 1999 ) found that hopanoids carry out the map in procaryotes that steroid alcohols perform in eucaryotes cells. From looking at consequences, one time tetrahydroxybacteriohopaneglycolipid was added to the membrane there was an ordering consequence. There have been some surveies that have been done, to happen the effects of hopanoid on cell membrane fluidness. Last twelvemonth research was carried out mensurating the consequence of diplopterol ( Hopanoid ) , on cell membrane fluidness. The consequences were stamp downing as it showed no positive or negative correlativity, bespeaking that there is no consequence on the membrane fluidness ; despite the fact the sample was purified ( fig 4.0.1 ) .
Diplopterol is a hopanoid that is found in procaryotes cell, but it is non the most abundant hopanoid. The extended hopanoids such as bacteriohopane and its derived functions could itself order the membrane ( Kannenberg and Poralla, 1999 ) .
Diplopterol is similar to cholesterol in the construction, but comparing to the extended hopanoids, the extended hopanoids are really similar. Cholesterol has hydrocarbon tail that is bonded to a five C ring. This allows the molecule to divider between the phospholipid dress suits so that the membrane would non be able to travel ( Halling et al. , 2008 ) . But diplopterol does non hold hydrocarbon concatenation and possibly that is the ground why there was no alteration in fluidness ( fig 4.0.2 ) .
To mensurate the membrane fluidness, steady-state fluorescence polarization was used. 1,6-Diphenyl-1,3,5-hexatriene ( DPH ) was the fluorescent investigation that was used to place any alteration in the membrane fluidness. The investigation dividers into the membrane bilayer mediate the hydrocarbon dress suits and one time exited from the beam of polarised visible radiation, it emits fluorescence which is detected ( fig 4.0.3 ) . This technique is utile in placing any alterations in the membrane fluidness giving an anisotropy value. If there is strong signal in the emitted fluorescence, this states that the motion of the investigation is restricted therefore bespeaking a decrease in membrane fluidness. If there is an addition in the emitted fluorescence this indicates there is a decrease in the mobility of the investigation therefore a lessening in the fluidness of the membrane. So as anisotropy additions, the membrane fluidness lessenings and if anisotropy decreases the membrane fluidness additions ( Shinitzky and Barenholz, 1978 ) .
4.1. Preparation of the Liposomes
In order to utilize the right lipid concentration for the liposome suspension in all the experiments, trials were done to demo, 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 lessening from 0.050mg/ml to 0.100mg/ml in anisotropy, and so it seems to hold a plateau part from 0.100mg/ml to 0.200mg/ml were the anisotropy value stayed changeless. From 0.200mg/ml to 0.500mg/ml the anisotropy value so decreased one time once more. Theoretically as the lipid concentration additions this should non impact anisotropy. The lessening from 0.050mg/ml to 0.100mg/ml could be due to dispersing, otherwise known as Raman Scattering. It was C. V. Raman that found, visible radiation is scattered from a molecule, and the photon that is scattered has the same energy as the visible radiation emitted. At low concentration of the lipoids, the sprinkling is caused by the aqueous environment in the cuvette. This causes intervention in the frequence and wavelength, demoing a high anisotropy value ( Banwell & A ; McCash, 1994 ) . From 0.200mg/ml to 0.500mg/ml the anisotropy value decreased. This is caused by light dispersing by the addition in lipid concentration. The concentration of the lipoid in the cuvette is high and the visible radiation emitted is scattered at different angles alternatively of the light passing through the solution to the sensor ( Banwell & A ; McCash, 1994 ) . At 0.100mg/ml to 0.200mg/ml the anisotropy values are changeless, demoing plateau part. It is within this part there is no Raman Scattering, and the visible radiation emitted is non scattered by the molecules in the cuvette ( Banwell & A ; McCash, 1994 ) . As there is no interface from the outside environment this consequence is dependable and the concentration of the lipoid 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 dissolver for which betulinic acid was dissolved in. Before trials that could be done, to see if betulinic acid ordered the membrane, experiments needed to be conducted to see whether DMSO affected the membrane entirely. Figure 3.2.1, shows a bead in anisotropy value as the volume of DMSO increased. There was bead from 50Aµl to 100Aµl by 0.014, bespeaking that the membrane going more fluid. But this lessening in anisotropy is little and will non impact the overall consequence one time betulinic acid it added to the membrane.
Betulinic acid dividers in the cell membrane, because in the aqueous solution, it is thermodynamically unfavorable. The construction is similar to cholesterin, which could explicate figure 3.2.3, demoing an addition in anisotropy value by 58 % . As the concentration of betulinic acid increases the membrane becomes more ordered. Looking at the construction of betulinic acid ( fig.1.2.1. ) there are two polar terminal groups at opposite terminals ; the polar hydroxyl ( OH ) group and a carboxyl ( COOH ) group. The carboxyl group are more polar so the hydroxyl group, and is likely to infix itself to the caput group of the phospholipid and the hydroxyl group will lie along side with the phospholipid tails. This could explicate why betulinic acid as the same belongingss as cholesterin in telling the membrane.
Experiments with cholesterin and betulinic acid reduced the membrane fluidness more efficaciously so betulinic acid entirely.
Cholesterol was added to the liposome suspension, at a ratio of 5:1, which was changeless throughout the whole experiment. Figure 3.2.4 shows that there is a higher lessening in membrane fluidness one time betulinic acid is added. Comparing to calculate 3.2.3, where it was merely betulinic acid that was added to the liposome suspension, the cholesterin and betulinic acid showed an addition in telling of the membrane, but non by much. There was merely a 28 % addition in anisotropy in figure 3.2.4 as the concentration of betulinic acid additions ; comparing to the betulinic acid alone it was a batch higher at 58 % .
Figure 3.2.4 shows the add-on of betulinic acid to a membrane that is already contains cholesterin at a 5:1 ratio. It is good understood that cholesterin stabilises membrane, cut downing cell membrane fluidness ( Ikonen, 2008 ) and at a ratio of 5:1, the membrane is tightly packed and ready ordered, doing it hard for betulinic acid to divider in to the membrane. The add-on of betulinic acid hence did non do much of an impact on membrane fluidness has thought.
It would be interesting to see if betulinic acid was added foremost at a 5:1 ratio, so cholesterin at different concentrations. But this causes jobs as cholesterin dissolves in trichloromethane and methyl alcohol at a 2:1 ratio. Chloroform is a nonionic compound ; therefore doing it non-miscible with H2O. If trichloromethane was added to the liposome suspension, this will interrupt the formation of the liposome and give false consequences.
4.3. Tetrahydroxybacteriohopaneglycolipid Measurements
Thin bed chromatography was preformed to see whether tetrahydroxybacteriohopaneglycolipid contained any drosss. The consequences from figure 3.3.1 showed one topographic point on the TLC home base. The Rf value was calculated to be 0.26, utilizing the equation in subdivision 3. The Rf value for tetrahydroxybacteriohopaneglycolipid, that was suggested by Dr Gunther Muth was to be 0.26, which relates to the topographic point that was on the TLC home base, taking to decision that the sample was pure.
For the experiments mensurating the anisotropy ; methanol was the dissolver, were tetrahydroxybacteriohopaneglycolipid was dissolves in, as recommended by the hopanoid provider ( Dr Gunther Muth, University of Tubingen ) . Methanol was so tested to see if it had any consequence on the membrane fluidness. Figure 3.3.2 shows a bead in anisotropy as volumes of methyl alcohol additions. Methanol increases the membrane fluidness, but the affect was non big plenty to do any interface, so the experiments with tetrahydroxybacteriohopaneglycolipid carried on.
Figure 3.3.3 shows the affect tetrahydroxybacteriohopaneglycolipid has on the membrane fluidness. As the concentration of tetrahydroxybacteriohopaneglycolipid additions the anisotropy value drops from 0.121 to 0.106, diminishing by 15 % . This was unexpected as it indicates that the membrane is going less ordered as the concentration of the hopanoid additions. The stock solution was 0.5mM doing the maximal concentration from figure 3.3.3, 0.0125mM. This gives a ratio of hopanoid: lipid molecules of 100:1.
This was investigated farther utilizing another method, were the hopanoid was incorporated in to the Personal computer, and so it was subjected to oxygen free N ( OFN ) before the buffer was added.
The alternate method will demo any alterations in anisotropy depending on the ratio of hopanoid: lipid molecules. The graph below shows a control with no hopanoids and besides a comparative consequence with hopanoids, were a liposome suspension was made that contained a ratio of hopanoid: lipid molecules of 100:1, similar to the maximal concentration from figure 3.3.3. From looking at the graph below, there is no alteration in the anisotropy value.
This experiment was so performed once more, but the ratio of hopanoid: a lipid molecule of 200:1.
The consequences above showed no difference in anisotropy value, intending there is no alteration in the fluidness of the membrane. This makes sense because presenting a compound that has a similar construction to cholesterol should non increase the fluidness of the membrane ; besides that there literature did non back up this determination. But figure 3.3.3 shows a lessening in anisotropy, this could be explained by methyl alcohol could hold caused the membrane to go more unstable. At really low concentration of hopanoid and high volumes of methyl alcohol this could be the chief ground why there was a lessening in figure 3.3.3.
Figure 3.3.3, 4.2.1 and 4.2.2 readings showed, at low concentrations tetrahydroxybacteriohopaneglycolipid had no consequence on the membrane fluidness.
Another stock solution was made were the concentration was 1.8mM. Figure 3.3.4 shows there is no alteration on the membrane fluidness, 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 cholesterin: lipid molecule of 30:1, there would be a little ordination affect.
Figure 3.3.5 showed a positive correlativity in anisotropy value as the concentration of tetrahydroxybacteriohopaneglycolipid additions. It increased by 7 % , were the maximal concentration of tetrahydroxybacteriohopaneglycolipid is 0.115mM giving a ratio of hopanoid: lipid molecules of 15:1. This showed that at a concentration runing from 0.045mM to 0.115mM causes the membrane to go more ordered.
This was investigated farther utilizing an alternate method, were Alternatively of mensurating 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 subdivision 2.5.
Figures 3.3.6 shows an addition of 18 % in anisotropy. This provides grounds that tetrahydroxybacteriohopaneglycolipid orders the membrane if the ratio of hopanoid: lipid molecules is 10:1.
Another experiment was conducted to see whether there is farther sweetening in telling the membrane at the ratio of 5:1. Figure 3.3.7 showed an addition of 28 % compared to the control. Figures 3.3.6 and 3.3.7 both showed that tetrahydroxybacteriohopaneglycolipid reduces the membrane fluidness. It besides 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 construction of tetrahydroxybacteriohopaneglycolipid ( fig. 1.2.1 ) . The theory has stated that tetrahydroxybacteriohopaneglycolipid contains similar belongingss to cholesterol in telling the membrane. Tetrahydroxybacteriohopaneglycolipid ‘s construction is similar to cholesterol as both have a hydrocarbon concatenation and besides they both contain a ring construction that stabilises the whole molecule. Tetrahydroxybacteriohopaneglycolipid contain an isopropyl concatenation were three hydroxyl ( OH ) groups are bonded covalently on C 32, 33 and 34. These polar caput groups are likely to organize H bonds with the phospholipid caput groups and the environing aqueous environment. To be able to organize the H bond, the hydroxyl ( OH ) group on the hopanoid must be in a I? conformation, confronting the C=O group on the caput of the phospholipid. The cyclo-hexane rings at the terminal of the molecule will tie in with the phospholipid dress suits, cut downing their motion. This prevents the hydrocarbon concatenation to revolve 120A° clockwise or anticlockwise organizing a crick otherwise known as gauche conformation. Introducing an amphiphilic molecule such as cholesterin, betulinic acid and tetrahydroxybacteriohopaneglycolipid this reduces the ability of the phospholipid molecule to organize a crick in the concatenation ( Whitening et al. , 2000 ) .
An experiment was done utilizing cholesterin with the same method above. Figure 4.2.3 shows the anisotropy value increased by 41 % , at cholesterin: lipid molecule ratio of 5:1. Compared to the hopanoid 5:1 ratio, cholesterin stabilises the membrane more efficaciously so the hopanoid.
This can be explained, were most eukaryote cells are composed of phosphatidylcholine forming liposomes, most prokaryote membranes are chiefly composed of phosphatidylethanolamine and phosphatidylglycerol. These phospholipids are able to organize upside-down micelles were hydrophilic caputs points inwards ( Gurr et al, 2002 ) . This difference could explicate why tetrahydroxybacteriohopaneglycolipid did n’t demo every bit much as an telling consequence as cholesterin did. It may be the difference in composing of the caput group of the phospholipids, why tetrahydroxybacteriohopaneglycolipid did n’t hold the same consequence. Another fact to indicate out is that tetrahydroxybacteriohopaneglycolipid contains a glycolipid which is hydrophilic at the terminal of the isopropyl concatenation. Possibly it is this fond regard, that possibly the ground why tetrahydroxybacteriohopaneglycolipid does n’t partition into the membrane bilayer every bit easy as cholesterin.
Surveies have reported that tetrahydroxybacteriohopaneglycolipid has the same map as cholesterin in the cell membrane ( Poralla, Kannenberg et Al. 1980 ) . They studied the bacteriums Bacillus acidocaldarius, which was alone in that it grows at pH 3 and 65A°C. They found that 15 % of the lipoids contain tetrahydroxybacteriohopaneglycolipid that might hold contributed to the thermophilic and acidophilous belongingss in Bacillus acidocaldarius. This would do sense, because cut downing the permeableness of the membrane will cut down the inactive diffusion of the protons and other solutes.
Betulinic acid has already been shown that it has cytotoxic belongingss. This in itself can bring on programmed cell death in malignant neoplastic disease cells ( Gopal et al. , 2005 ) . Future work can be done, to see how betulinic acid would be able to aim cells, chiefly malignant neoplastic disease cells. A job that is associated with betulinic acid is that it ‘s really harmful to the organic structure. It has the belongingss to bring on programmed cell death in malignant neoplastic disease cells, but besides other normal cells will be affected if non decently monitored. But before betulinic acid can be used as a drug, trials need to be done to demo if the environment such as temperature and pH has an consequence on betulinic acid ( Immordino et al. , 2006 ) . Like hopanoids, betulinic acid has derived functions that have besides been found in workss. It would be interesting to see if derived functions such as betulin and dihydrobetuline acid can impact the membrane.
There were three more hopanoids that was gained from Dr Gunther Muth. It would be really interesting to see and compare hopanoids that have an isopropyl concatenation and hopanoids that do n’t on membrane fluidness. Hopene the simplest hopanoid, that could be compare to tetrahydroxybacteriohopane the simplest hopanoid with the isopropyl concatenation. This so will bespeak if it is the hydroxyl group on the isopropyl concatenation and the concatenation itself, which causes the stabilization belongings.
Hopanoids with different groups attached could besides impact the cell membrane such as aminotrihydroxybacteriophan which contains a hydrocarbon concatenation, but besides has a glucosamine group bonded. It would be interesting to see if other derived functions like aminotrihydroxybacteriophan will do a more or less ordering consequence to the membrane.
The belongings of bracing the membrane could give the hopanoid a cytotoxic nature ; altering the fluidness and the permeableness of the cell membrane could interrupt the conveyance of indispensable molecules through the cell membranes.
The lipid fluidness can be measured in a figure of ways. Some are dearly-won but more sensitiveness and some are inexpensive with less sensitiveness. Nuclear magnetic resonance is a powerful experimental technique that can foreground any facets of the lipid bilayer alteration. The phospholipids will be treated with 31P or 2H and NMR spectroscopy gives the information on alterations in the construction and kineticss of the phospholipids of the theoretical account lipid membrane. If there are any alterations in the membrane fluidness, it will demo extremums, and if the extremums are farther apart, the membrane is less fluidised. Another method that can be used is colorimetric enzyme check. This method does non mensurate the fluidness of the membrane, but measure the contents in the membrane. For illustration it can be used to see whether molecule dividers into the membrane, and so the fluidness can be measured utilizing steady province fluorescence polarization.