In the Laboratory
Identification and Quantitation of Plasma Membrane Components: A Biochemical Experiment for Lipid Investigations
W
Susan Keys Biology/Chemistry Department, Springfield College, Springfield, MA 01109;
[email protected] Rationale This biochemistry exercise is designed for upper-level undergraduate science students who have some, but not necessarily extensive, laboratory experience. Students learn important laboratory techniques as they investigate the lipid composition of erythrocyte plasma membranes. The techniques, thin-layer chromatography (TLC) and spectrophotometry, are simple and do not require expensive equipment or involve the use of large volumes of toxic reagents. The exercise requires two 2-hour laboratory periods. In the first period, students isolate erythrocytes from blood and extract lipids from these cells. (This step can be done by the instructor ahead of time if only one lab period is to be used.) In the second period, they determine the identity of the major lipid components and quantify the phospholipid content of their samples. The sequence of steps in the exercise allows students to experience the progressive continuity from cell isolation to molecular identification and quantification that is central to many biochemical investigations. In addition, the successful completion of microscale procedures teaches students the importance of concentration, precision, and attention to detail in the laboratory. Undergraduate training in laboratory microtechniques is invaluable for students aspiring to graduate programs related to biochemistry or careers in biotechnology. Background Biological membranes are composed of a lipid bilayer and associated proteins. The lipid bilayer contains phospholipids (PL), cholesterol, and glycolipids. It provides the structural framework of the membrane and acts as a barrier to water-soluble molecules and ions. All living cells and most intracellular organelles are surrounded by membranes. Membranes from different cell types and even different organelles from the same cell differ in lipid composition. In fact, the diverse functions of different cells and various organelles depend on the design of their individual membranes (1). Erythrocytes are often used to investigate membranes because they comprise a plasma membrane surrounding cytosol devoid of any intracellular membranous organelles. Because there are no contaminating intracellular membranes, any lipid components extracted from these cells are derived from the plasma membrane. The composition of phospholipids varies considerably in erythrocyte membranes from different species (2). It even varies between the two faces of the bilayer. In most mammalian erythrocytes, the extracellular face of the plasma membrane bilayer contains phosphatidylcholine, sphingomyelin, and glycolipids, while phosphatidylethanolamine and phosphatidylserine are found on the cytosolic face (3). Cholesterol is distributed equally in both layers (2).
All membrane lipids can be separated and identified using thin layer chromatography. This technique is fast, easy, and relatively inexpensive, and it provides good resolution with the appropriate solvent system. A phosphorus assay using spectrophotometry allows quantification of the total phospholipid content. Procedure
Separation of Erythrocytes Sheep’s blood (Carolina Biological Supply #F6-82-8890) is used as an inexpensive, safe source of erythrocytes. The blood must be centrifuged to separate the cellular elements from the plasma. A tabletop clinical centrifuge is sufficient for this application. Centrifugation at 1000 × g for 5 minutes yields a pellet of erythrocytes overlaid by a pale layer of leukocytes and platelets and a supernatant of blood plasma. Separation of Membrane Lipid Components Dissolve the pellet of erythrocytes in a mixture of chloroform/methanol (2:1 by volume) and 0.02 N HCl (4 ). Centrifuge at 1000 × g for 10 minutes. Lipids will be contained in the lower chloroform phase of the extract. Proteins (mainly cytoplasmic hemoglobin) will form a red layer at the interface between the chloroform layer and the upper aqueous layer. The separation procedure yields approximately 2 mL of chloroform extract containing all the lipids derived from the erythrocyte plasma membranes. An alternative procedure is to make erythrocyte ghosts by placing erythrocytes in a hypotonic solution, pellet the membranes, and then treat with chloroform/methanol as described above. Identification of Lipid Components by T LC Membrane lipid components are separated by thin-layer chromatography and identified by comparing marker plates with experimental plates. Lipid TLC employs silica gel, acidwashed during manufacture. TLC plates may be obtained from Whatman, Clifton, NJ (#4860-320). Marker plates are made by applying known lipid standards (cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingomyelin) and measuring the R f of each standard. All standards are at concentrations of 25–40 nmol/µL. Experimental plates are prepared with the mixture of lipids extracted from the erythrocyte plasma membranes. After separation of the components of the mixture, the R f of each component is measured and compared with the R f of the standards. Samples are applied with a capillary pipet or micropipet as small dots at the origin. As little as 5 µL of the known samples may be applied to the marker plate. The lipid mixture
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
1463
In the Laboratory
Quantification of Phospholipids by Phosphorus Assay To quantify the phospholipids contained in the membrane samples, students must first construct a standard curve using samples of varying known amounts of phosphorus. Phosphatidylcholine (2.5 mg/mL chloroform) can be used as a standard. The representative curve (Fig. 1) includes nine data points representing 0–67 nmol of phosphatidylcholine (PC). The spectrophotometric assay used in this exercise is accurate for small samples (2–70 nmol of phosphorus). Each group of students assays several volumes of the PC to construct a standard curve (ranging from 3–67 nmol of PC). They also assay five different volumes of the mixture of lipids extracted from erythrocyte membranes. Each sample is placed in an acid-cleaned, dry test tube and oxidized by heating in 10% MgNO3⭈6H2O in 95% ethanol. This step takes about 10 minutes. The oxidation produces carbon dioxide and water (which are lost to the atmosphere) and inorganic phosphate. The remaining inorganic phosphate is measured spectrophotometrically after the addition of 10% ammonium molybdate in 4 N HCl and 0.2% malachite green (6 ). After plotting a standard curve using the known concentrations of phosphatidylcholine, students estimate the phospholipid concentration of the membrane samples. Since the amount of lipid may vary between extractions, students can compare relative amounts of phospholipid in several aliquots of lipid extract to test the accuracy of their standard curve. These samples can be processed simultaneously with the PC standards to save time and eliminate variations in technique. Each individual lipid component can be quantified to determine the relative proportions of the various phospholipids in the membrane extract, but this procedure requires an additional lab period. Individual lipid components are separated by TLC as above. The location of each lipid on the experimental plate is determined by comparing a stained marker plate and an unstained experimental plate. The areas containing the individual phospholipids are marked and scraped from the plate. Individual lipids are removed from the silica gel by dissolving in chloroform–methanol. The phosphorus assay is conducted on the dissolved lipids to determine the amount of each species. Hazards TLC procedures should be performed in a fume hood as the solvent contains chloroform. Test tubes are heated over a Bunsen burner during the oxidation step of the phosphorus assay. Tubes should be pointed away from students (preferably in a fume hood) as the contents may splatter as carbon dioxide and water are released.
1464
1.2
1.0
Absorbance 660 nm
from the erythrocyte membranes is less concentrated than the known samples, so a larger volume must be applied to the experimental plate. Samples of 20–50 µL show all major lipid components if the application spot is kept as small as possible. The solvent is a mixture of chloroform–methanol–acetic acid–0.9% NaCl (75:45:3:1 by volume) (5). Run time is approximately 15 minutes. The lipid bands can be visualized by staining with iodine.
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
50
60
70
Phosphorus / nmol Figure 1. Representative standard curve to be used to quantify phosphorus obtained from membrane lipid extract.
Equipment Needed Tabletop centrifuge 90° C oven TLC chambers Micropipettors or capillary pipets (1–50 µL) Results and Discussion Erythrocytes from different species have different phospholipid profiles. Students compare the experimentally determined phospholipid profile of sheep’s blood with the phospholipid content of human blood. They see that sheep’s blood contains no phosphatidylcholine, while human blood contains a significant portion of PC. They discuss the possibility that a lack of PC confers resistance to some snake venoms (phospholipases specific for PC) in ruminants. The reported lipid composition of human erythrocyte plasma membranes is 23% cholesterol, 18% phosphatidylethanolamine, 18% sphingomyelin, 17% phosphatidylcholine, 7% phosphatidylserine, 16% minor components (7), but the proportions of individual lipids vary with the animal species. Apparently, phosphatidylcholine and sphingomyelin are interchangeable in the outer leaflet of the bilayer, so the proportions of these two phospholipids vary considerably among species. If blood from a ruminant (sheep, for instance) is used, PC will be completely absent and there will be a much larger band of sphingomyelin (8). If blood from other mammalian species (rat, dog, horse, pig, human) is analyzed, all five of the compounds used as standards will be present in the red blood cell lipids. The composition of erythrocyte plasma membranes is unusual because of the relatively large proportion of cholesterol. Polar lipids like phospholipids and glycosphingolipids can be separated in solvent systems based on chloroform–methanol with dilute acetic acid (9). Neutral lipids like cholesterol travel with or near the solvent front. Students are able to identify all the major lipid components using this solvent system by comparing the R f values of the
Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
In the Laboratory Table 1. R f Values of Lipid Standards Rf Value
Lipid
Expected
Student Result a
Cholesterol
.94
.94 ± .05
Sphingomyelin
.11
.13 ± .02
Phosphatidylcholine
.17
.16 ± .03
Phosphatidylserine
.20
.21 ± .03
Phosphatidylethanolamine
.51
.59 ± .04
NOTE: Determined by TLC using a solvent composed of chloroform–methanol–acetic acid–0.9% NaCl (75:45:3:1 by volume). aMean of 6 values ± SE.
Table 2. Results of Spectrophotometric Assay of Phospholipids in Sheep Er ythrocytes Lipid Aliquot/µL
A6 6 0
PL/nmol
0.03
0
50
0.15
5
100
0.27
10
200
0.50
21
350
0.85
38
500
1.00
53
0.(reagent blank)
known standards with the R f values of the lipids separated from the membrane extract by thin-layer chromatography. Expected R f values of the standards in this solvent system (10) are shown in Table 1. These values are compared with representative student data compiled from six groups of two students each and presented as the mean ± standard error. Students confirm the relative accuracy of their standard curve (Fig. 1) by determining the phosphorus content of a series of aliquots of the erythrocyte lipid extract (Table 2). If
the curve is constructed accurately and if the each aliquot contains 2–70 nmol of phosphorus, the extrapolated PL content of different amounts of extract will be proportional to their volumes. The representative data presented in Table 2 show, for instance, that doubling the volume of an aliquot also doubles the amount of phosphorus. Students learn several useful techniques in this series of exercises. After isolating a particular population of cells from whole blood, they learn how to separate lipid-soluble and water-soluble cellular components. They then separate and identify individual lipids from a mixture using TLC. Finally, they construct and use a standard concentration curve to estimate the phosphorus content of unknown samples. WSupplemental
Material
Supplemental material for this article is available in this issue of JCE Online. Literature Cited 1. Unwin, N.; Henderson, R. Sci. Am. 1984, 250 (2), 78–84. 2. Marchesi, V. T.; Furthmayr, H. Annu. Rev. Biochem. 1976, 45, 667–698. 3. Zwaal, R. F. A.; Roelofsen, B.; Colley, C. M. Biochim. Biophys. Acta 1973, 300, 159–182. 4. Folch, J.; Lees, M.; Sloan-Stanley, G. H. J. Biol. Chem. 1957, 226, 497–509. 5. Allan, D.; Cockcroft, S. J. Lipid Res. 1982, 23, 1373–1374. 6. Buss, J.; Stull, J. T. Methods Enzymol. 1983, 99, 7–14. 7. Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of the Cell, 3rd. ed.; Garland: London, 1994; p 482. 8. Turner, J. C.; Anderson, H. M.; Gandal, C. P. Biochim. Biophys. Acta 1958, 30, 130. 9. Broekhuyse, R. M. Clin. Chim. Acta 1974, 52, 53. 10. Kates, M. In Techniques of Lipidology: Isolation, Analysis, and Identification of Lipids, 2nd ed.; Burdon, R. H.; van Knippenberg, P. H., Eds.; Elsevier: Amsterdam, 1986; p 384.
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
1465
