From DSM Manual, courtesy Hans-Juergen Busse.
Extraction and analysis of respiratory lipoquinones.
A number of methods have been described for the extraction of respiratory lipoquinones from microorganisms. These include the use of acetone, mixtures of chlorform:methanol (2:1 v/v) or hexane:methanol (1:2 v/v). Extraction is usually carried out using freeze dried cell material, although freshly harvested or frozen cell material is used by some groups. In order to avoid loss of lipoquinone material it is avisable to carry out the extraction under nitrogen atmosphere, away from strong sunlight, and at temperatures below 35-40 C. The method described is routinely used int he author's laboratory, and permits the subsequent extraction of polar lipids from the same biomass.
1. 100 mg of freeze dried cell material is weighed into small screw cap bottles (10 ml capacity, brown glass) fitted with teflon-coated septa.
2. A small stirring bar is made from a 1 cm long piece of paper clip an also placed in the bottle.
3. To the cell material in the bottle is now added 3 ml of hexane:methanol (1:2 v/v).
4. Briefly gas the cell suspension with nitrogen and seal the bottle with the screw cap, making sure that the teflon-coated side seals against the mouth of the bottle.
5. The bottle is placed on a magnetic stirrer (about 10 such bottles can be placed on the stirrer at once) and left to stir for 30 min.
6. After 30 min stirring under nitrogen, the extracted material is placed in an ice bath until the hexane and methanol phases begin to separate.
7. Ice cold hexane (1 ml) is then added to give a methanol:hexane (1:1 v/v) biphasic mixture. Separation of the layers is enhanced by centrifuging the suspension in glass centrifuge tubes at 3000 rpm for 5 min.
8. The upper, hexane, phase is removed with a pasteur pipet and placed in a small glass tube (4-5 ml capacity).
9. The methanol phase is further extracted by the addition of cold hexane (2 ml) and 0.3% NaCl (2 ml) to give a 1:1:1 (v/v/v) ratio of hexane, methanol, and 0.3% NaCl. Centrifuge as above.
10. The upper hexane layer is then removed and added to the first hexane fraction.
11. The hexane phase is concentrated under a stream of nitrogen to give a final volume of about 0.5 ml, and the extract applied as a thin line to the lower edge of a silica gel plate (containing a fluorescent indicator F254).
12. The plates are developed in hexane:tert-butylmethylether (9:1 v/v) until the solvent front reaches the upper edge of the plate (20-30 min).
13. The developed plate is examined under UV light (254 nm) briefly to locate respiratory lipoquinones, which appear as dark bands against a greenish background. Menaquinones have an Rf value of about 0.7, while ubiquinones have an Rf of about 0.4.
14. The lipoquinone material is removed from the silica gel by scraping the appropriate area from the thin layer plate and placing it in a narrow bore tube (or pasteur pipet) fitted with a frit or glass wool plug. After tapping the tube to settle the silica gel, the lipoquinone material is eluted by passing 2x 0.5 ml of hexane:methanol (1:2 v/v) through the column, the eluant being collected in a small glass bottle (1.5 ml capacity). The purified lipoquinones may be recovered into hexane by cooling the mixture until phase separation occurs, followed by the addition of a few drops of 0.3% NaCl and about 0.3 ml of cold hexane. The upper, hexane phase containing the lipoquinones, is transferred to a second small 1.5 ml glass bottle, and may be stored at -20 C for analysis by UV spectroscopy, HPLC, or mass spectroscopy.
15. Further analysis of the lipoquinones is carried out by evaporating the hexane phase. HPLC analysis of the lipoquinones is carried out by dissolving the material in 100-200 ul of the HPLC solvent. UV spectroscopy is carried out by dissolving eh lipoquinone in a suitable volume (usually 0.5 - 1 ml) of spectroscopic grade hexane, ethanol, or (more commonly) iso-octane. Mass spectra of lipoquinones are recorded on material dissolved in the minimum quantity of a suitable solvent, such as methanol.
UV Spectroscopy
UV Spectra of respiratory lipoquinones give information on the nature of the quinone nucleus. UV spectra may be recorded in hexane, ethanol or iso-octane (the latter being the most frequently used). Spectra are recorded between 200 nm and 500 nm in quartz cuvettes. Menaquinone type lipoquinones (naphthoquinones) give four maxima between 230 nm and 280 nm, while ubiquinone type lipoquinones (benzoquionones) give two maxima lying very close together between 260 nm and 280 nm.
Polar Lipid Extraction
Polar lipids are generally extracted from dry cell material using chloroform:methanol:0.3% NaCl (1:2:0.8 v/v/v). This may be carried out by adding 9.5 ml of this mixture to 100 mg of freeze dried cells, or by adding a suitable amount of chloroform, methanol and 0.3% NaCl to the cell material, or to the aqueous methanolic phase remaining from the lipoquinone extraction.
1. The aqueous methanolic phase (4 ml total volume), together with the cell material from the lipoquinone analysis, is diluted with 5.5 ml of Chloroform:Methanol (2.5:3.0 v/v) to give a chloroform, methanol, 0.3% NaCl (1:2:0.8 v/v/v) mixture.
2. The mixture is placed in a 15 ml bottle with a teflon lined screw cap (check that the magnetic stirrer is in the bottle), gassed briefly with nitrogen, sealed and heated for 15 min at 80 C (with occasional shaking).
3. Allow the mixture to cool to room temperature on a magnetic stirrer. Check that the mixture is homogeneous, the presence of excess hexane will cause phase separation and may be overcome by adding a small amount of methanol until a homogeneous mixture is obtained.
4. The cell debris is removed by centrifugation in glass centrifuge tubes, at 3000 rpm for 5-10 min, and the supernatant decanted into 5 ml of chloroform:0.3% NaCl (1:1 v/v). The latter mixture (2.5 ml chloroform and 2.5 ml of 0.3% NaCl) may be placed in the extraction buffer bottles after they have been washed with distilled water.
5. After brief mixing, the biphasic mixture is centrifuged in glass centrifuge tubes (3000 rpm for 5 min) and the lower, chloroform phase collected with a pasteur pipet. Take care not to remove the flocculent protein layer or the aqueous phase.
6. The chloroform phase, containing the polar lipids is taken to dryness under a stream of nitrogen (when using a heating block or a water bath to accelerate drying, do not exceed 40 C).
7. The dried lipid material is then re-dissolved in 250 ul of chloroform:methanol (2:1 v/v) and transferred toa small glass bottle or ampule and may be stored for periods of several months at temperatures of -20 C or lower.
8. The lipid solution is used to spot thin layer plates; the lipids being placed in the bottom left-hand corner of each plate (Fig 4-A). Glass or aluminum plates are used because many detection reactions require the plates to be heated.
Run the first dimension with the spotted lipid on the lower right hand corner. After drying, run the plates in the second dimension with the lipid in the lower left hand corner.
9. Develop the plates in two dimensions using, in the first dimension chloroform:methanol:water (65:25:4 v/v/v), and in the second dimension, chloroform:methanol:acetic acid:water (80:12:15:4 v/v/v/v). Between the two dimensions, the plates should be dried at room temperature for about 20-30 min.
10. Lipid functional groups are identified using spray reagents specific for phosphate (Zinzadze), alpha-glycols (periodate-Schiff), and sugars (alpha-naphthol/H2SO4, anisaldehyde/H2SO4), free amino groups (ninhydrin), quaternary nitrogen (Dragendorff), and primary and secondary amines (chlorine and starch/iodide). Identification of the various lipids is carried out on the basis of staining reaction and Rf values. All spraying must be carried out in a fume hood using sufficient ventilation, since all lipid spray reagents are toxic.
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