Demonstration pubs.acs.org/jchemeduc
Osmotic Stressing, Membrane Leakage, and Fluorescence: An Introductory Biochemistry Demonstration Kalani J. Seu* Department of Chemistry, Earlham College, 801 National Road West, Richmond, Indiana 47374, United States ABSTRACT: A fluorescence demonstration is described that incorporates several fundamental aspects of an introductory biochemistry course. A variation of a known leakage assay is utilized to prepare vesicles containing a quenched fluorophore. The vesicles are exposed to several osmotic environments ranging from isotonic to hypotonic. The degree of vesicle swelling, partial rupture or bursting, and subsequent release and dequenching of the encapsulated fluorophore can be observed. This demonstration introduces students to fluorescence, fluorescence quenching, lipid membrane dynamics, membrane permeability, and osmotic pressure. Students are also introduced to the lipid extrusion process and are able to observe the separation of compounds via size exclusion chromatography. This demonstration has the flexibility to be expanded to laboratory experiments that allow students to investigate a variety of membrane properties. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Biophysical Chemistry, Hands-On Learning/Manipulatives, Fluorescence Spectroscopy, Chromatography, Lipids, Membranes
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INTRODUCTION Fluorescence has become a very prevalent and powerful tool in biochemical and biophysical research. Although most textbooks do not have dedicated chapters for fluorescence and fluorescence-based methods, it is a topic covered in most biochemistry courses. At Earlham College, the current Biochemistry course has a dedicated section on fluorescence and fluorescence techniques that are covered after units on lipids and biological membranes and transport. While there are many demonstrations and activities that provide a way for students to visualize fluorescence,1−7 many of these simply demonstrate the fluorescence process. However, an important aspect of teaching is the ability to help students make mental connections; this includes making connections between various chapters of course material, as well as to practical applications. The demonstration described herein presents fluorescence properties in the context of a practical application in biochemical and biophysical research. Moreover, it incorporates and links many concepts and aspects of chemistry and biochemistry, including cell membrane properties and osmotic pressure. This demonstration exposes students to a variety of techniques and concepts, and emphasizes the importance and power of fluorescence as a biochemical tool.
with the prepared CF-encapsulated lipid vesicles in solutions ranging from near-isotonic to hypotonic is shown in Figure 1.
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DESCRIPTION OF THE DEMONSTRATION Lipids are a major component of cell membranes, and lipid vesicles have been used extensively as a simple model membrane system.8−11 As with real cell membranes, the membranes of lipid vesicles are semipermeable, flexible, and self-healing. In this demonstration, lipid vesicles are first made in a high salt solution containing a high concentration of the fluorophore carboxyfluorescein (CF). A series of five test tubes © XXXX American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Test tubes, each with 100 μL of CF-containing lipid vesicles with 2000 μL of (from left to right) 700, 525, 350, 175, and 0 mM NaCl viewed in a dimmed environment using long wave UV excitation. Vesicles in the test tubes vary from near-isotonic (far left) to a hypotonic environment (far right).
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DOI: 10.1021/acs.jchemed.5b00023 J. Chem. Educ. XXXX, XXX, XXX−XXX
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MO). Triton X-100 was purchased from Fisher Scientific. An ultraviolet (UV) Mineralight Lamp from UCP, LLC was used for excitation of CF, primarily at 366 nm.
The high concentration of CF results in dynamic/collisional quenching between photoexcited state CF molecules returning them to the ground state without photon emission; thus, while trapped inside the vesicles, no fluorescence is observed (Figure 1, far left tube). Placing the vesicles in a similar high salt (isotonic) solution results in little to no net water movement across the vesicle membrane, whereas placing them in a low salt (hypotonic) solution results in a net inward movement of water across the vesicle membrane. In the hypotonic solution, the lipid vesicles swell as a result of the outward pressure on the membrane by the water influx and, if the pressure is great enough, eventually burst, releasing the entrapped CF. The CF released from the vesicles is diluted and becomes dequenched, resulting in fluorescence emission of the free fluorophores (Figure 1, far right tube). In an intermediate salt solution, in which the osmotic pressure is less, pores form in the vesicles, allowing the solutions to reach osmotic equilibrium, and the membranes reseal rather than burst. In this case, a fraction of the dye is released and dequenched, while some remains quenched within the resealed vesicles, resulting in an intermediate amount of fluorescence observed. The fact that the lipid vesicles do not burst under all salt conditions demonstrates that membranes are fluid, dynamic, and semipermeable. Some of these aspects may be explored qualitatively by simple additions to the demonstration. For example, changing the membrane composition to 80% POPC/ 20% POPE12 results in a visible decrease in membrane leakage at intermediate osmotic pressures. This change can be attributed to differences in the POPE shape factor,11 as well as increased membrane viscosity resulting from headgroup hydrogen bonding.13,14 Detergent effects15 and the effects of organic molecules, such as ethanol and methanol,16,17 on membrane stability can also be demonstrated and discussed. The ideas underlying this visual demonstration of osmotically induced membrane leakage can be investigated more thoroughly in a laboratory setting. Although that goes beyond the scope of this demonstration paper, it should be noted that quantitative fluorimetry measurements have been used to explore a wide range of such effects11,18 and such studies are certainly suitable for undergraduate laboratory investigations.
Lipid Vesicle Formation Using Extrusion
POPC in chloroform (5 μmol) is added to a 50 mL roundbottom flask. Chloroform is removed in a fume hood under a gentle flow of nitrogen gas, leaving a thin lipid film in the round-bottom. The flask is left under vacuum for a minimum of 45 min to ensure that all of the chloroform is removed. The lipid film is solubilized in 1 mL of CF buffer (100 mM CF, 700 mM NaCl, 50 mM phosphate, pH = 7.4) and vortexed for 1−2 min to ensure that all of the lipid film is removed from the round-bottom. The solution is extruded through a 50 nm polycarbonate membrane using the Mini-Extruder as described in the vendor-provided instructions. The extruded suspension of 50 nm vesicles is transferred to an Eppendorf tube. Size Exclusion Chromatography
The vesicles containing CF are separated from free CF fluorophores using size exclusion chromatography. A few grams of Sephadex G-50 are solubilized in an excess of a near-isoelectric buffer (700 mM NaCl, 50 mM phosphate, pH = 7.4) overnight. Approximately 40 mL of the solubilized Sephadex G-50 is added to a 2 × 30 cm glass chromatography column. The extruded vesicles are added to the column and separated using several column volumes of the near-isoelectric buffer as the mobile phase. The vesicles can be identified as a light orange band that will elute from the column before the free CF (Figure 2). The separated vesicles containing the encapsulated CF (1−2 mL) are collected in Eppendorf tubes for use in the demonstration. Size exclusion chromatography of the vesicles takes approximately 20−30 min and can be done as part of the demonstration or beforehand, if necessary. Students are often introduced to liquid and thin layer chromatography techniques, but few students have the opportunity to observe size exclusion chromatography. If time permits, this is a good opportunity for students to experience the use of size exclusion chromatography. Vesicle Osmotic Stressing and Fluorescence Leakage
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CF-containing vesicles (100 μL) are added to each of five test tubes. Phosphate buffer (50 mM pH = 7.4) (2 mL) containing varying concentrations of sodium chloride (700, 525, 350, 175, and 0 mM NaCl) is added to each test tube and mixed. The resulting mixtures are placed under a UV light (366 nm) in a dimmed room for observation. Any vesicle leakage or rupturing occurs quickly after mixing and fluorescence should be visible immediately. If desired, a detergent, such as Triton X-100, can be added to a sixth tube to demonstrate the complete disruption of the vesicles and total dequenching of CF.
PREPARING THE DEMONSTRATION Preparation for this demonstration can be done in parts and ahead of time to varying degrees. If the goal of the demonstration is to show osmotic stressing and the release of the encapsulated, quenched fluorophore, then the preparation of the vesicles, including the purification by size exclusion chromatography, can be done ahead of time; demonstrating the osmotic stressing of the vesicles takes only a few minutes. If other aspects of the demonstration will be emphasized, such as bilayer formation, the entire process including vesicle preparation, size exclusion, and osmotic stressing can be done in 60−90 min.
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HAZARDS Lipids purchased from Avanti Polar Lipids, Inc. may come dissolved in chloroform, which is a dangerous substance and a suspected carcinogen. Appropriate precautions should be taken when working with chloroform. Work done with chloroform should be conducted by an instructor with the appropriate knowledge of the dangers of this substance. UV light can damage the eyes and skin. Do not look at the UV light source or point it in the direction of the audience. Appropriate eye and personal protective equipment should be worn when using the UV light. Triton X-100 can be harmful if swallowed. It is also an eye irritant and is toxic to aquatic life. Gloves and eye
Materials
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in chloroform (10 mg/mL) and a Mini-Extruder Set containing a mini-extruder, two 1 mL syringes, 50 nm polycarbonate membranes, and filter supports were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 5(6)-Carboxyfluorescein (CF), sodium chloride, Sephadex G-50, sodium phosphate dibasic heptahydrate, and sodium phosphate monobasic monohydrate were purchased from Sigma-Aldrich (St. Louis, B
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composition of the vesicles can also be altered to explore the effects of different lipid compositions on membrane stability.10,11 Previous publications on the uses of lipid vesicles in undergraduate biochemistry laboratory experiments have focused on the formation and shape19,20 and passive transport properties21 of lipid vesicles. This demonstration introduces an alternative fluorescence-based method to explore lipid membrane dynamics, membrane permeability, and osmosis/osmotic pressure effects. This demonstration, thus, ties together several sections of material that students are learning throughout a course, and has the additional advantage of emphasizing fluorescence as an important biochemical tool.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS K.J.S. thanks Katie Giger, Paul Ogren, and Mark Stocksdale for their suggestions and comments. Thanks also to Earlham College and the students from the 2009−2014 Earlham College Biochemistry courses for enthusiastically embracing this demonstration.
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Figure 2. Size exclusion chromatography of 50 nm vesicle suspension. The lipid vesicles containing encapsulated CF (band near the middle of the column) are separated from the CF in free solution (dark orange band) using Sephadex G-50 and a quasi-isoelectric buffer as the eluent. The CF-containing lipid vesicles elute before the free CF molecules due to their larger size.
REFERENCES
(1) Feigl, F.; Heisig, G. B. Some New Demonstrations on Fluorescence. J. Chem. Educ. 1952, 29 (4), 192−194. (2) Bozzelli, J. W. A Fluorescence Lecture Demonstration. J. Chem. Educ. 1982, 59 (9), 787−788. (3) Burrows, H. D. A Convenient Lecture Demonstration of Fluorescence. J. Chem. Educ. 1983, 60 (3), 228. (4) Blitz, J. P.; Sheeran, D. J.; Becker, T. L. Classroom Demonstrations of Concepts in Molecular Fluorescence. J. Chem. Educ. 2006, 83 (5), 758−760. (5) MacCormac, A.; O’Brien, E.; O’Kennedy, R. Classroom Activity Connections: Lessons from Fluorescence. J. Chem. Educ. 2010, 87 (7), 685−686. (6) Clarke, R. J.; Oprysa, A. Fluorescence and Light Scattering. J. Chem. Educ. 2004, 81 (5), 705−707. (7) O’Reilly, J. E. Fluorescence Experiments with Quinine. J. Chem. Educ. 1975, 52 (9), 610−612. (8) Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S. W.; Zarghami, N.; Hanifehpour, Y.; Samiei, M.; Kouhi, M.; Nejati-Koshki, K. Liposome: Classification, Preparation, and Applications. Nanoscale Res. Lett. 2013, 8 (1), 102−110. (9) Torchilin, V. P. Recent Advances with Liposomes as Pharmaceutical Carriers. Nat. Rev. Drug Discovery 2005, 4 (2), 145− 160. (10) Shoemaker, S. D.; Vanderlick, T. K. Stress-Induced Leakage from Phospholipid Vesicles: Effect of Membrane Composition. Ind. Eng. Chem. Res. 2002, 41 (3), 324−329. (11) Hull, M. C.; Sauer, D. B.; Hovis, J. S. Influence of Lipid Chemistry on the Osmotic Response of Cell Membranes: Effect of Non-Bilayer Forming Lipid. J. Phys. Chem. B 2004, 108 (40), 15890− 15895. (12) POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine); POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). (13) Seu, K. J.; Cambrea, L. R.; Everly, R. M.; Hovis, J. S. Influence of Lipid Chemistry on Membrane Fluidity: Tail and Headgroup Interactions. Biophys. J. 2006, 91 (10), 3727−3735.
protection should be worn when using this substance. All other chemicals and substances used in this demonstration, including CF, phosphate buffer salts, Sephadex G-50, and sodium chloride, are not considered to be hazardous substances according to MSDS sheets.
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SUMMARY This demonstration utilizes CF-encapsulated lipid vesicles, in a high ionic environment subjected to osmotic stress by the addition of solutions of varying ionic concentrations. As the ionic strength decreases, the vesicles swell and ultimately burst, releasing and dequenching the encapsulated CF, visualized using a UV light, allowing students to observe the results of osmotic stress. Techniques such as lipid vesicle extrusion and size exclusion chromatography can also be demonstrated. The demonstration described requires no specialty instrumentation and the Mini-Extruder Kit from Avanti Polar Lipids, Inc. is reasonably affordable ($470; enough for at least 25 demonstrations). This demonstration can be expanded upon and may, with the appropriate resources, be turned into a laboratory experiment. For example, students can explore the effects of various detergents, organic solvents, or natural compounds on the osmotic fragility of the vesicles. Additionally, the released CF fluorophore can be quantified using a fluorimeter and students can calculate the osmotic pressure associated with each solution, as well as the maximum osmotic pressure of the membrane before rupturing. The lipid C
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(14) Pitman, M. C.; Suits, F.; Gawrisch, K.; Feller, S. E. Molecular Dynamics Investigation of Dynamical Properties of Phosphatidylethanolamine Lipid Bilayers. J. Chem. Phys. 2005, 122 (24), No. 244715. (15) Lichtenberg, D.; Ahyayauch, H.; Alonso, A.; Goni, F. M. Detergent Solubilization of Lipid Bilayers: A Balance of Driving Forces. Trends Biochem. Sci. 2013, 38 (2), 85−93. (16) Klemm, W. R. Biological Water and Its Role in the Effects of Alcohol. Alcohol (N. Y., NY, U. S.) 1998, 15 (3), 249−267. (17) Patra, M.; Salonen, E.; Terama, E.; Vattulainen, I.; Faller, R.; Lee, B. W.; Holopainen, J.; Karttunen, M. Under the Influence of Alcohol: The Effect of Ethanol and Methanol on Lipid Bilayers. Biophys. J. 2006, 90 (4), 1121−1135. (18) Logisz, C. S.; Hovis, J. S. Effect of Salt Concentration on Membrane Lysis Pressure. Biochim. Biophys. Acta, Biomembr. 2005, 1717 (2), 104−108. (19) Jakubowski, H. V.; Penas, M.; Saunders, K. The Study of Lipid Aggregates in Aqueous Solution: Formation and Properties of Liposomes with an Encapsulated Metallochromic Dye. J. Chem. Educ. 1994, 71 (4), 347−349. (20) Del Bianco, C.; Torino, D.; Mansy, S. S. Vesicle Stability and Dynamics: An Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2014, 91 (8), 1228−1231. (21) Paula, S. An Introduction to Passive Ion Transport across Model Lipid Membranes for Undergraduate Students: Proton Permeation Measurements in Liposomes. J. Chem. Educ. 2014, 91 (1), 145−148.
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DOI: 10.1021/acs.jchemed.5b00023 J. Chem. Educ. XXXX, XXX, XXX−XXX
