Osmotic Stressing, Membrane Leakage, and Fluorescence: An Introductory Biochemistry Demonstration

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.

iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and Lipids Easily Extracted from Chicken Eggs

“… Shape changes of vesicles, including prolates and oblates, can be promoted just by enabling the outer medium to evaporate . The inner water of vesicles permeates to equilibrate the osmolarity gradient between the inside and outside by osmosis and permeability processes Budding events can be observed by externally adding different lipophilic molecules to prolate vesicles …”

Liposome permeability probed by laser light scattering

“…For example, in the last 40 years, only eight papers (e.g., [1][2][3][4]) concerning lipids and membranes were published in this journal, five of which described undergraduate laboratory projects. Over this same time period, eight more lipid/membrane papers (e.g., [5][6][7][8][9][10]) were published in the Journal of Chemical Education, six of which presented laboratory projects. As a comparison, just in the year 2017 the Journal of Chemical Education Laboratory Section included 15 papers on proteins and enzymes, 3 on sugars and carbohydrates, 3 on DNA/ RNA/nucleic acids, and none on lipids.…”

“…Physical characteristics of the membrane bilayer are controlled by the lipid composition of the membrane [4,6], including fatty acid length and unsaturation, and head group size and charge [10,12,14]. Three main physical characteristics of the bilayer influence biological function: fluidity [3,4,7,10,15,16], permeability [3,5,8,9,17], and protein interaction [2,10,16,18]. Fluidity is a key parameter, directly altering membrane permeability [16], and indirectly influencing protein interactions [2,10,16,18].…”

“…For example, raising the temperature increases membrane fluidity, which decreases lipid packing density and causes the membrane to expand, which in turn increases permeability. Previously published laboratory projects have assayed transmembrane permeability using fluorophores and UV chromophores . In addition, it is also possible to use simple UV–vis light scattering to follow membrane expansion or contraction .…”

Size Exclusion Chromatography: A Teaching Aid for Physical Chemistry

“…Size exclusion chromatography (SEC), the premier method for characterizing macromolecules in terms of molecular mass and molecular mass distribution, gives educators another approach for introducing polymer science into undergraduate chemistry programs. ,, A literature search of this Journal on SEC as a pedagogical approach for studying macromolecules has revealed a number of papers on the use of this technique in the undergraduate curriculum; − however, there are none that address SEC theory from a thermodynamic perspective.…”