3 Биологические мембраны. Обмен веществом (1160072), страница 7
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A commonly used extractant is a mixture of chloroform, methanol, and water, initially in proportions that are miscible,producing a single phase (1:2:0.8, v/v/v). After homogenizing tissue inthis solvent to extract all lipids, more water is added to the resultingextract, and it separates into two phases, methanol/water (top phase)and chloroform (bottom phase). The lipids remain in the chloroform,and more polar molecules (proteins, sugars) partition into the polarphase of methanol/water (Fig.
9-21).Chapter 9 LipidsFigure 9-21 Some common procedures used in theextraction, separation, and identification of cellularlipids. (a) Tissue is homogenized in a chloroform/methanol/water mixture, which on addition ofwater and removal of unextractable sediment bycentrifugation yields two phases. Different typesof extracted lipids in the chloroform phase may beseparated by (b) adsorption chromatography on acolumn of silica gel, through which solvents of increasing polarity are passed, or (c) thin-layer chromatography (TLC), in which lipids are carried by arising solvent front, less polar lipids traveling farther than more polar or charged lipids.
TLC withappropriate solvents also can be used to separateindividual lipid species from a single class; for example, the charged lipids phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol are easilyseparated by TLC.
For the determination of fattyacid composition, a lipid fraction containing esterlinked fatty acids is (d) transesterified in a warmaqueous solution of NaOH and methanol, producinga mixture of fatty acyl methyl esters, which arethen (e) separated on the basis of chain length anddegree of saturation by gas-liquid chromatography.Precise determination of molecular mass, by massspectroscopy (not shown), allows unambiguous identification of individual lipids. The lipid is ionizedand volatilized by heat and the resulting molecularion is passed through an electromagnetic field,which deflects ions to a degree dependent on theirsize.
By comparison with standard ions of knownmolecular mass, the mass of the unknown molecular ion is determined with such great accuracy thatthe structure of the lipid can be deduced.263Tissuehomogenized inchloroform/methanol/water(a)i-waterMethanol/waterChloroform(b)AdsorptionchromatographyThin-layerchromatographyII IIAdsorption Chromatography SeparatesLipids of Different PolarityThe complex mixture of tissue lipids can be fractionated further bychromatographic procedures based on the different polarities of eachclass of lipid.
In adsorption chromatography (Fig. 9-21), an insoluble,polar material such as silica gel (a form of silicic acid, Si(OH)4), ispacked into a long, thin glass column, and the lipid mixture (in chloroform solution) is applied to the top of the column. The polar lipids bindtightly to the polar silicic acid, but the neutral lipids pass directlythrough the column and emerge in the first chloroform wash. The polarlipids are then eluted, in order of increasing polarity, by washing thecolumn with solvents of progressively higher polarity.
Uncharged butpolar lipids (cerebrosides, for example) are eluted with acetone, andvery polar or charged lipids (such as glycerophospholipids) are elutedwith methanol.Thin-layer chromatography on silicic acid (Fig. 9-21) employs thesame principle. A thin layer of silica gel (silicic acid) is spread onto aglass plate, to which it adheres. A small sample of lipids dissolved inchloroform is applied near one edge of the plate, which is dipped in a1 2 3 4 5 6 7 8 9<Neutral Polar Chargedlipids lipids lipids(d)I NaOHAnethanolTFatty acyl methyl estersGas-liquidchromatographyoCoUElution time264Part II Structure and Catalysisshallow container of an organic solvent within a closed chamber saturated with the solvent vapor.
As the solvent rises on the plate by capillary action, it carries lipids with it. The less polar lipids move farthest,as they have less tendency to bind to the polar silicic acid. The lipidscan be detected after their separation by spraying the plate with a dye(rhodamine), which fluoresces when associated with lipids, or by exposing the plate to iodine fumes. Iodine reacts with the double bonds infatty acids, giving the lipids that contain them a yellow or brown color.For subsequent analysis, regions containing separated lipids can bescraped from the plate and the lipids recovered by extraction with anorganic solvent.Gas-Liquid Chromatography Resolves Mixturesof Volatile Lipid DerivativesGas-liquid chromatography separates volatile components of a mixtureaccording to their relative tendencies to dissolve in the inert materialpacked in the chromatography column, and to volatilize and movethrough the column, carried by a current of an inert gas such as helium.
Some lipids are naturally volatile, but most must first be derivatized to increase their volatility (that is, lower their boiling point). Forthe analysis of the fatty acids present in a sample of phospholipids, thelipids are first heated in a methanol/HCl or methanol/NaOH mixture,which converts fatty acids esterified to glycerol into their methyl esters(transesterification). These fatty acyl methyl esters are then loadedonto the gas-liquid chromatography column, and the column is heatedto volatilize the compounds. Those fatty acyl esters most soluble in thecolumn material partition into (dissolve in) that material; those lesssoluble are carried by the stream of helium and emerge first from thecolumn (Fig.
9-21). The order of elution depends on the nature of thesolid adsorbant in the column, and on the boiling point of the components of the lipid mixture. Using these techniques, mixtures of fattyacids with various chain lengths and various degrees of unsaturationcan be completely resolved.Specific Hydrolysis Aids in Determination of Lipid StructureCertain classes of lipids are susceptible to degradation under specificconditions.
For example, all ester-linked fatty acids in triacylglycerols,phospholipids, and sterol esters are released by mild acid or alkalinetreatment, and somewhat harsher hydrolysis conditions releaseamide-bound fatty acids from sphingolipids. Enzymes that specificallyhydrolyze certain lipids are also useful in the determination of lipidstructure. Phospholipases A, C, and D (see Fig. 9-12) each split specificbonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example,releases a water-soluble phosphoryl alcohol (phosphocholine fromphosphatidylcholine) and a chloroform-soluble diacylglycerol, each ofwhich can be characterized separately to determine the structure ofthe intact phospholipid.
The combination of specific hydrolysis withcharacterization of the products by thin-layer chromatography or gasliquid chromatography often allows determination of the structure of alipid. To establish unambiguously the length of a hydrocarbon chain, orthe position of double bonds, mass spectral analysis of lipids or theirvolatile derivatives is invaluable.Chapter 9 Lipids265SummaryLipids are water-insoluble components of cells thatcan be extracted by nonpolar solvents. Some lipidsserve as structural components of membranes andothers as storage forms of fuel.
Fatty acids, whichprovide the hydrocarbon components of lipids, usually have an even number (12 to 24) of carbonatoms and may be saturated or unsaturated; unsaturated fatty acids have double bonds in the cisconfiguration. In most unsaturated fatty acids, onedouble bond is at the A9 position (between C-9 andC-10).Triacylglycerols contain three fatty acid molecules esterified to the three hydroxyl groups ofglycerol. Simple triacylglycerols contain only onetype of fatty acid; mixed triacylglycerols contain atleast two different types. Triacylglycerols are primarily storage fats; they are present in many typesof foods.The polar lipids, which have polar heads andnonpolar tails, are major components of membranes. The most abundant are the glycerophospholipids, which contain two fatty acid moleculesesterified to two hydroxyl groups of glycerol, and asecond alcohol, the head group, esterified to thethird hydroxyl of glycerol via a phosphodiesterbond.
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