Electron Transport Chain in Respiratory Complex I

Electron Transport mountain filament in Respiratory composite IIntroductionE real cosmosness depends on energy to survive, in order to adduce an organized verbalise, homeostasis, with metabolism and oppositewise biochemical reactions. Energy is generated in a look of diametrical ways depending on the organism. Mammals create energy by dint of the disruption of organic molecules, a good deal(prenominal) as carbohydrates, proteins and lipids, that yields other compounds that drives cellular processes. One such compound is adenosine triphosphate (Adenosine triphosphate) an essential energy-carrying molecule that is synthesised by respiration by a series of enzyme protein conglomeratees put together in the mitochondria. intricate I (NADHcoenzyme Q oxidoreductase) is wholeness of those essential protein aimd embedded in mammalian mitochondria. NADH produced by the Krebs tricarboxylic acid rung and - oxidization of fatty acids, is change to initiate the mechanist ic nerve way of Complex I, ultimately reducing ubiquin unrivaled and establish proton-motive stuff crossways the inner membrane of the mitochondria. It is this proton gradient that will support the generation of adenosine triphosphate from adenosine triphosphate synthase and other stub processes.Signifi markt research has been conducted on Complex I, particularly from Bovine heart mitochondria, however to date many aspects of this enzyme is lock poorly understood due to its building interlinking structural arrangement and ways undertaken. To retrace its mechanism, will eventu each(prenominal)y chair to a greater understanding in the role of Complex I in many diseases and dysfunctions.MitochondriaMitochondria ar junior-grade sub-cellular organelles compound in a series of processes generally with its role in the respiratory placement. Occupying al about 10% to 30% of cell volumes of sizings ranging between 0.75 and 3m, the unique shape of a mitochondrion allows the process to take place, with its tell apart structural feature organism a double membrane.1 These both membranes argon crumbled by the intermembrane space and overall enclose the central matrix. Whereas the let outmost membrane is inundated by porins to assist the movement of solutes of about 12 kDa or less the inner membrane is impermeable to solutes simply presents the deification env straighten outment for the establishment of an electrochemical proton gradient, by the presence of numerous protein thickeninges. redundant compartments of the organelle include the cristae and the mitochondrial matrix, which comprises a plethora of enzymes involved in ATP metabolism.Additionally, a range of studies devote likewise indicated the ability of mitochondria to form changing ne cardinalrks of interconnected tubules that regulates the cell structure to adapt to its specific function when required. As a result, during disruption of such networks, cellular dysfunction can occur, track to a flake of neural related syndromes such as Parkinsons and Alzheimers.2,3 a boldness from the primary role of energy metabolism, the mitochondria overly power other core cellular functions such as apoptosis, calcium handling and the formation of press out sulfur assembles.The sideline sections discuss the primary(prenominal) enzymes involved in the negatron transport set up that lead to the generation of ATP, particularly respiratory knotty I, which will be the main focus of this thesis.Respiratory ComplexesComplex IIAlso cognize as succinate ubiquinone oxidoreductase, complex II is a 120 kDa enzyme consisting of tetrad nuclear-encoded subunits which be arranged in two disciplines.4 It is this distinctive arrangement which allows this enzyme to oxidise succinate to fumarate which is coupled to the production of ubiquinol finished the lessening of ubiquinone in the mitochondrial inner membrane. time it is involved with cofactors, this enzyme complex does not nowadays go to the proton motive force in order to establish a chemical gradient.4,5Succinate+ Q Fumarate + QH2 equivalence 1 2 of the enzymes subunits SdhA and SdhB form a deliquescent, succinate dehydrogenase subcomplex and forms the succinate/fumarate fecundation site whereas SdhB contains collar iron- treat clusters which argon embedded to the mitochondrial membrane by the rest SdhC and SdhD subunits.4 These latter subunits contain a heam group and ubiqionone stick to sites. When a flavin dinucleotide, which is ligated to SdhA, it oxidises succinate, the negatrons produced in this process atomic number 18 passed down with the iron-sulphur clusters. The electrons after allow the lessening of ubiquinone to ubiquinol.6,7Complex deuce-aceComplex III or ubiquinolcytochrome c oxidoreductase is an 240 kDa enzyme which is made up of 11 subunits. Its structure comprises of two ubiquinone attach sites Qo, present towards the mitochondrial membrane, catalyses the oxidati on of ubiquinol to ubiquinone and Qi, present towards the matrix, catalyses the reduction of ubiquinone to ubiquinol.8,9Complexes I and II produces ubiquinol from the reduction of ubiquinone, which binds to the Qo site on complex III. During this process, an electron is passed on the iron-sulfur cluster reducing it and moving it towards cytochrome c1 and cytochrome c resulting in a conformational variety show. The change ca social functions a second electron to be communicatered done another pathway organise of cytochromes bL and bH towards to Qi bind site, in where it allows the formation of a semiquinone anion by dint of the reduction of an already bound ubiquinone. Parallel to this, a second quinol is oxidised at Qo allowing the electron to be transferencered through the starting pathway of Rieske iron-sulphur cluster and cytochrome c1 and the second electron follows the second pathway mentioned above to Qi, reducing the semiquinone anion to ubiquinol.10 The oxidation at Qo releases cardinal protons into the inter-membrane space of the mitochondria and the reduction at Qi results in the uptake of two protons from the matrix which ar transferred into the inter-membrane space during ubiquinol oxidation. This complete cycle allows the reduction of two cytochrome c molecules.9QH2 + 2 cyt c3+ + 2H+in Q + 2 cyt c2+ + 4H+outEquation 2Oxidation and reduction cycles in Complex III results in the movement of four protons into the inter-membrane space maintains the proton motive force utilize by ATP synthase to synthesise ATP.8Complex IVComplex IV, alike cognize as cytochrome c oxidase, is an enzyme, which comprises of 13 subunits, of which three are encoded by the mitochondrial genome. The enzyme catalyses the oxidation of cytochrome c which leads to the reduction of group O to water allowing the translocation of four protons crosswise the mitochondrial inner membrane.11,12The oxidation of cytochrome c produces electrons that are transferred to an a ctive site where molecular oxygen is minify. This reduction producing water releases free energy required for the pumping of four protons from the matrix of the mitochondria into its inner-membrane space. This movement of protons is facilitated through two known proton channels the K-channel passes two protons for the reduction of oxygen and the D-channel allows the movement of newly translocated protons.13O2 + 4 cyt c2+ + 8H+in 2 H2O + 4 cyt c3+ + 4H+outEquation 3The translocated protons and the reduction of oxygen to water allows ATP synthase to generate ATP as this contributes to the proton motive force similar to Complex III.Complex VPrimarily known as ATP Synthase, this enzyme complex operates by utilising the proton chemical gradient completed in the intermembrane space by the preceding complexes, to drive the synthesis of ATP from adenosine diphosphate and inorganic Phosphate. With an average size of 580 kDa, the enzyme is self-possessed of 16 subunits organised in two hydrophobic and hydrophilic estates the hydrophobic domain forms a proton semiconductive pore through the inner membrane while the hydrophilic domain, containing three copies of and subunits, spreads into the matrix. The two domains are linked by an asymmetric central walk and a computer peripheral stalk, which acts as a stator to prevent the F1 domain rotating freely during catalysis. The interfaces between the two subunits forms the screen sites for automatic data processing and inorganic Phosphate. 14,15ADP + P+ nH+in ATP + nH+outEquation 4Complex IComplex I, is the first and largest enzyme involved the electron transfer chain of the mitochondrion. Alternatively known as NADHubiquinone oxidoreductase, its primary role is to oxidise NADH and ultimately reduce ubiquinone.16NADH + H+ + Q + 4H+in NAD+ + QH2 + 4H+outEquation 5Just like the other protein complexes, the potential energy released from the redox reaction within the complex, translocates four protons across the i nner membrane for every(prenominal) molecule of oxidized NADH and removes two additive protons from the matrix for the reduction of quinone. The processes contribute to the overall electrochemical gradient which is to be apply by ATP synthase to synthesise ATP.17StructureTo date, complex I has been found in a variety of species, including many prokaryotes. The complex I from bovine heart mitochondria is primarily utilise in studies due to its close sequential identity with the human being complex I enzyme. The mammalian complex I is one of the most complex and largest enzymes known, with a combined mass of 980 kDA and sedate of at to the lowest degree 45 polar polypeptide subunits with 14 strictly conserved core subunits that are necessary for function and also common across the among all known complex I.16 The significance of the additional subunits in complex I among dissimilar species thus far remain a riddle. It is known some be involved in protection against reactive oxygen species generation and some are required needed for proper assembly and stableness of the enzyme.16,18As ascertained by single-particle electron microscopy (EM) for both bacterial and mitochondrial enzymes, the determined structure of the enzyme closely resembles to an L shape, with septet hydrophobic core subunits that constitutes the membrane tail domain and cardinal hydrophilic core subunits that constitutes peripheral (hydrophilic) arm domain project into the mitochondrial matrix which is known as the catalytic domain as it includes all redox centres and dorsum site while the membrane domain consists loosely of hydrophobic subunits. 16While the full structure of the eukaryotic complex is not tranquil puff up characterised, in 2006, Sazanov group successfully report structure of the hydrophilic domain of complex I from Thermus thermophiles bacteria.20The Peripheral section of complex IThe peripheral arm of the complex is composed of seven individual subunits, that together, houses the NADH-oxidizing dehydrogenase module, which provides electron input into a noncovalently-bound flavin mononucleotide (FMN) molecule. The molecule sequentially transfers the electron to a chain of nine iron-sulphur (Fe-S) clusters, eight of which are found in the bovine enzyme. Additionally, the hydrophilic arm also comprises of a Q-module, which conducts electrons to the quinone-binding site for quinol production. 16,20 wholly of theseWithin the respiratory chain complexes, at that place are three different types of Fe-S clusters, two of which, are found in complex I Two binuclear 2Fe-2S and six tetranuclear 4Fe-4S clusters.As the name suggests, the binuclear clusters are composed of two iron atoms that function as bridged by two acid-labile sulphur atoms. Each iron atom is also coordinated by an additional two sulphur atoms found on the surrounding cysteine residues from the protein complex. In the tetranuclear Fe-S clusters, four iron atoms and four sulphur a toms are arranged in a cube with each iron atom also ligated to sulphur cysteine-residue on the surrounding protein, similar to binuclear Fe-S.22 receivable to their conformational arrangements and redox capabilities provided by the iron atom, these clusters act as electron transfer agents or also known as ferrodoxins. The detection of these clusters can be achieved by EPR (electron paramagnetic resonance) which is successfully achieved in many studies. However, out of the two binuclear and six tetranuclear iron-sulfur clusters found in complex I, only two binuclear and four tetranuclear clusters are EPR active.22Figure 1. structures of the iron-sulphur clusters found in complex I.As previously mentioned, seven of the eight clusters, form a 95 -long extensive chain at a time from the flavin site to the quinone binding site on the interface of the membrane domain. Even though the distances between these chains may seem cold apart, as much as 14 , distances are close sufficient to allow electron transfer to occur.23,24However, the presence of the eight cluster is still not well understood. Cluster 2Fe24 found on the opposite attitude of the Flavin site, is believed not to be involved in electron transfer pathway. While it was just a theory with no evidence, it has been proposed that this additional cluster functions as an electron store that accepts an electron from the flavosemiquinone species preventing the generation of reactive oxygen species during enzyme turnover.24 membrane Domain of complex IThe membrane domain comprises the proton-translocating module which catalyses proton transport. With the exception of subunit ND1 and the quinone binding site, found on the interface of the peripheral arm, the membrane domain functions totally independently from the two arms of complex I.Within the membrane domain, there are four structural subunits that return been identified to be possibly involved with proton translocation these include subunits ND2, ND4 and ND5. There is also an additional transporter which believed to be either ND1, ND6 or ND4L. Each believed to be transporting one proton per catalytic cycle. Each individual subunits are composed of charged residues and helices that creates half-channels that allow the passage of proton to occur. The membrane structure is also held together by a long -helix chain that spans across its intact length. Its feature is to maintain and support the integrity of the membrane domain.26Overall Mechanism of complex IThe mammalian complex I includes 45 known proteins, out of which 14 core subunits comprises of both hydrophilic and hydrophobic domains as explained above.16The mechanism through the electron transfer chain starts with a Flavin mononucleotide (FMN) molecule which is non-covalently bound to the 51kDa subunit through hydrogen bonds at the top of the hydrophilic domain. FMN molecule oxidises NADH prima(p) to the reduction of iron-sulphur clusters (Fe-S) which transfers electrons from Flavin to the quinone-binding site 51. This electron transfer distorts the conformation of the protein through changes in its redox state leading to alterations in pKa values of its side chains these alterations allows four hydrogen ions being pumped out of the mitochondrial matrix.24It is believed NADH gets oxidised to NAD+ through a hydride transfer avoiding the formation of the unstable NAD. Radical.24 This oxidation process occurs when the nicotinamide ring of the NADH lies above the flavin isoalloxazine system, allowing the electron donor hydride (C4 of the 27 nicotinamide ring) and acceptor (N5 of the flavin) to roll in the hay within 3.5 of each other and transfer electrons.28As explained above, NADH oxidation leads to transfer of electrons through seven iron-sulphur clusters chain between Flavin and quinone reduction binding site in the membrane.20 It is the final Fe-S cluster that donates the electrons to the bound ubiquinone substrate which is believed to be accessed t hrough an entry point in the membrane to the binding site.21These iron-sulphur clusters are best detected apply a technique called electron paramagnetic resonance (EPR). Previous studies bear observed five decrease Fe-S clusters through EPR from Bovine compliex I reduced by NADH, and their spectra are equal N1b, N2, N3, N4 and N5.25 This technique will be advertize explained throughout this thesis.A much recent accept by Roessler et al. (2010) use EPR to understand the tunnelling electron transfer pathway through these clusters. Previous studies have already established EPR signals N1b, N2 and N3 are detected from 2Fe cluster in the 75 kDa subunit (position 2), and from 4Fe clusters in the PSST (position7) and 51 kDa subunits (position 1) respectively along the clusters chain due to interactions with ubisemiquinones and flavosemiquinone. As the other EPR signals have yet failed to be assigned to a particular cluster, Roessler et al. (2010) went on to use double electron-electr on resonance (DEER) spectroscopy to detect N4 and N5. Their results demonstrate that N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3).25The study propose an alternating energy potential profile for electron transfer along the chain between the actives sites, in B.taurus, which enhances the rate of a single electron travelling through the empty chain subsequently leading to more efficient energy conversion in complex I.25Followed by the iron-sulfur cluster is the site of quinone reduction. A study performed by Sazanov and Hinchliffe has identified a supposed binding site for the quinone precede group from T. thermophilus complex I hydrophilic domain between the 49 kDa and PSST subunits.20 This alleged site is close to the cluster where the ubiquinone substrate accepts electrons from the chain and it has also been acknowledged the 49 kDa and PSST subunits play an important role in quinone binding and catalysis.29Nevertheless, it is believed that additional hydrophobic subunits may also be involved in quinone binding and these are still being investigated.Even though the mechanism of NADH oxidation and ubiquinone reduction is comparatively well understood, how this oxidoreduction leads to quinone reduction and subsequent protons pumping across the mitochondrial membrane from complex I still remain a mystery. A number of theories for complex I mechanism have been proposed establish on the proton-pumping systems of the other mitochondrial respiratory complexes. These theories have been outlined belowA direct unification mechanism as demonstrated by complex IV through cytochrome c oxidase where the proton transfer is determined by a gating reaction occurring at the same time as the electron transfer reaction that started it.30An confirming coupling mechanism as seen in complex V (ATP synthase) explained previously. A study performed by Efremov et al., sug gests that within complex I, one proton is translocated by a directly coupled mechanism at the Fe-S clusters and the rest are go when quinone reduction drives conformational changes to the four-helix bundle of Nqo4 and of Nqo6 in complex I, subsequently poignant the C-terminal helix of Nqo12. The C-terminal has been identified by the authors running parallel to the membrane. The effect on this helix consequently leads to the other three helices to tilt which results in proton translocation.31A Q-cycle-like mechanism as represented by complex III where quinol is used as a carrier to transport protons across the mitochondrial membrane. A study completed by Dutton and co-workers suggested the complete reverse of this mechanism for complex I featuring the presence of two ubiquinone binding sites one facing the inter-membrane space, Qo, and the other facing the mitochondrial matrix, Qi. The quinone substrate would bind at Qi, and be reduced by one electron from a quinol already bound at Qo and another electron from the Fe-S cluster subsequently leading to two protons being taken up from the matrix while the formed semiquinone specie is still bound at Qo. Following the uptake of the protons, semiquinone is oxidised to ubiquinone.32 Nevertheless, further studies conducted have found no evidence of ubiquinol oxidation signifying complex I do not work through this mechanism.30,33While the first isolation of complex I from bovine heart mitochondria by Joe Hatefi et al occurred 40 days ago, information on its overall mechanism of action is still very limited particularly the mechanism of redox-proton coupling occurring in the membrane domain. To further understand this, new studies are being conducted to trap radical intermediates formed at the interface of the peripheral and membrane arm to establish the pathway that initiates proton translocation.Semiquinone radicalsSemiquinones are catalytic intermediates formed within complex I during the reduction of quinones at the quinone binding site and can exist in neutral or anionic form. Due to the presence of the unpaired electron, semiquinone intermediates can be studied using EPR spectroscopy.There are numerous pathways in which the formation of semiquinones can occur from quinone. The scheme below, proposed by Roessler and Hirst, illustrates the three main possible routes taken to obtain quinol.Pathways A and B involves with the generation of a neutral semiquinone radical specie based on the transferring of a proton and electron. On the other hand, pathway C which follows through pathway B involve with the generation of an anionic radical specie generated from an electron transfer. All pathways lead to formation of quinol by series of electron transfer and protons. The pathway shown in grey which occurs from the protonation of the neutral semiquinone radical specie will result in a 1-electron-2-centre bond which are energetically unstable.27Aside from one study, absolute majority of the studie s till date, have proved the existence of semiquinones by observing EPR signals using submitochondrial particles (SMPs). As the name suggests, these are inverted membrane vesicles housing the entire electron transport chain containing all enzyme complexes.34 However, since quinone cofactors are used by majority of the other complexes, distinguishing the semiquinone signals with each complex, has been far from successful.More recently, there has been a wave of research focusing on the identification of semiquinone radicals only when from complex I, however these have proved even more challenging as the organic intermediates produced very low intensity signals.Within complex I, there are two species of semiquinone that have been identified SQNf and SQNs.35,36 Based on their EPR properties, SQNf or fast restful semiquinones has been reported only during the presence of an established proton gradient across the membrane. On the other hand, SQNs or slow relaxing semiquinones, are not ef fected by proton gradient. The presence of two semiquinones has also lead to the possibility of complex I to contain two separate quinone binding sites Due to SQNf having a spin-spin interaction with Fe-S cluster N2, it is theorised that SQNf binding site is located close to the cluster at around 12 estimated distance, in contrast, SQNs binding site is suggested to be located around 30 from N2 cluster.22,25,37Within the complex, the SQNf is believed to be involved in proton pumping and its site aids the system by acting as bound co-factor site that facilitates the transfer of one electron from one site to another allowing the formation of a binding pocket for the SQNs in equilibrium with the ubiquinone pool of the membrane.22,25,32,35,38The presence of two separate quinone binding sites still remains a mystery and cannot be totally ruled out even though it has been suggested that SQNf and SQNs signals are detected from the same semiquinone species located from different sites or p resent in catalysis states.39A recent potential way of observing semiquinone intermediates via EPR is through the use of liposomes. Liposomes containing just Complex I or proteoliposomes, will facilitate the capture of semiqinone within its native environment and hopefully provide an penetration in the mechanism of Complex I and the binding of Q10.LiposomesLiposomes are global nanovesicles used in a variety of applications. Composed of a phospholipid bilayer, these clarified vesicles have an aqueous solution core surrounded by a hydrophobic membrane. Hydrophobic chemicals associate with the bilayer while the hydrophilic solutes dissolved in the core cannot readily pass through the bilayer essentially mimicking the cellular phospholipid bilayer. Due to these features, liposomes can be loaded both with hydrophobic or hydrophilic molecules and are excellent drug carriers or in this case house protein complexes. Liposomes are also not of course occurring and must be un pictorially g enerated using lipid extracts by aggregating them.40As liposomes are formed from naturally occurring lipids of low intrinsic toxicity, they are biodegradable and non-toxic. The functionality of liposomes is dependent based on three main factors. These include size, bilayer composition and liposome scratch properties.40Phospholipids are one the essential components in the formations of liposomes and can be divided into synthetic and natural phospholipids. They consist of two fatty acids hydrophobic chains linked to a hydrophilic (polar) head group, and they have either glycerol or sphingomyeline as the back bone. Having both hydrophobic and hydrophilic components, make phospholipids having amphipathic molecules.41 The diversity of the hydrophilic head group molecules and hydrophobic chains length allows the formation of different phospholipids which affects the surface charge and bilayer permeability of the liposomes.40The length and degree of saturation of the hydrocarbon acyl grou p chains determines the stability of the liposomal membrane, by affecting the temperature at which the membrane changes from a closely packed gel phase to a melted phase. The surface charge of the liposomes is determined by the charge of the lipid forming it which can be altered by modifying lipids with hydrophilic moieties to membrane bilayers.40Liposomes can be composed of naturally-derived phospholipids such as cholesterol, one of the commonly used lipids in liposome formation. It enhances the stability of the lipid bilayer and form highly ordered and rigid membrane with fluid like characteristics. Other phospholipids, synthetic and non-synthetic, can also be used for the formation of the liposomes such as pure surfactant components like DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).42Classifications of liposomesLiposomes are classified according to their morphological sizes and lamellarity, depending on their composition and method of formation.40Multilamellar vesicles (MLVs) consists some(prenominal) concentric phospholipid bilayers or lamellar ranging between 100nm to 20 m in size depending on the method of preparation. These large bilayers allow the integration of lipophilic molecules and proteins. olive-sized unilamellar vesicles (SUVs) single phospholipid bilayer and sized between 20 nm to 100nm. Ideal for encapsulation miniscule compounds and proteins.Large unilamellar vesicles (LUVs) single phospholipid bilayer with size ranging from 100 nm to 1 m. They are known to have larger aqueous core compared with or MLVs, reservation them suitable to expedient to load with numerous compounds.Oligolamellar vesicles (OLVs) vesicles similarly structured to MLVs but consists of anywhere between two and five phospholipid bilayers.Multivesicular liposomes (MVLs) When a large liposome vesicle similar in size to an MLV, enclose a group of liposomes, and so the subsequent vesicle is known as multivesicular liposome (MVL).Figure 1.40The veritable state o f research on liposomes have primarily been focusing on the face of drugs and other compounds to biological systems since it overcome challenges associated with reaching the target, making them very useful in the cosmetic and pharmaceutical industries.40Furthermore, it should be noted, some surfactant based phospholipids can mimic the biological systems helping construct important set systems for the research on enzymes and membranes. Many recent publications concerning liposomes have been focused on using this mimetic chemistry, which deals with models, mimicking cellular membrane to facilitate the research into their structures as well as the mechanisms both in vivo and in vitro.40Aims of ProjectThe reliable state of research on complex I remain mostly focused on the determination of the mechanism since only a subdivision has been found. Fully understanding will help solve many diseases and other complication caused by complex I.Whereas the mechanism of the reactions between NADH and iron sulphur clusters have been established, little is known about the mechanism of proton translocation as well as the role and existence of semiquinones that will lead into revealing more information into the function of the enzyme. The work described in the following records, using the best technique available, EPR, will aim to be using current studies of using liposomes to mimic cellular conditions, similar to the mitochondrial membrane, for complex I in order to obtain data regarding reduction of Q10 and proton translocation.MaterialsPreparation of Complex I from Bovine MitochondriaPreparation of Complex I proteoliposomes Stock solutions of 25 mgmL-1 of POPC in anaesthetise was transferred to a glass homogeniser with the required amount of ubiquinone-10 contained in chloroform. The chloroform was removed under Argon. An alternative approach is to remove under mindlessness using rotary evaporator. The resulting phospholipid film was resuspended in 675 L of buffer (10 mM Tris-SO4 (pH 7.5) and 50 mM KCl), and extruded 25 times through a Whatman 0.1 m pore membrane. The liposome mixture was solubilised with the addition of 160 L of octyl-glucoside from an aqueous 10% stock solution, sonicated for 10 min, and further incubated on ice for 10 min. The following steps were carried out at 4 C. 0.2 mg of AOX (50 L of 7.8 mgmL-1) and 0.2 mg of complex I (10 L of 20 mgmL-1) were added to the solubilised lipids and incubated for a further 10 min, followed by the addition of 100 L of SM2 Biobeads. The mixture wa