Biology Reference
In-Depth Information
of cholate or deoxycholate. The outer ring is stabilized by polar (
OH) interactions with
water. The charged carboxylate on the detergents further stabilizes the cylinder.
SDS (sodium dodecylsulfate) is by far the best known of a family of related anionic deter-
gents that bind to and denature proteins. Similar detergents can have different alkyl chain
lengths and different anionic head groups (e.g. sulfonate, carboxylate, phosphate etc.). Below
the CMC, SDS monomers bind to both membrane and cytosolic proteins in an equilibrium-
driven process. Binding is cooperative meaning the binding of a first SDS facilitates binding
of a second and so forth. Upon SDS binding, the protein structure is altered into rigid rods
whose length is proportional to its molecular weight. SDS binds to the protein chain at a ratio
of one SDS for every two amino acid residues. Since the SDS head is negatively charged, net
charge on the denatured protein is proportional to the protein's molecular weight. SDS-laden
proteins can then be separated in an electric field generated on a stable support. The process
is referred to as SDS-PAGE (polyacrylamide gel electrophoresis). The function of SDS in
membrane studies is therefore to determine the number and size of membrane proteins.
Since proteins are denatured by SDS, this detergent is of little value in reconstituting func-
tional proteins into membranes. SDS has many more applications outside the world of
science. It is a powerful surfactant that in high concentrations is the major ingredient in
many industrial cleaners and at low concentrations is found in toothpaste, shampoo, shaving
cream, bubble bath formulations, and even laxatives.
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B. MEMBRANE PROTEIN ISOLATION
Methodologies for the isolation and purification of integral membrane proteins have
always lagged far behind those for water-soluble cytosolic proteins. Awater-soluble protein
can often be precipitated by ammonium sulfate (salting-out) in relatively pure form. In sharp
contrast, integral membrane proteins must first be extracted from the membrane and come
attached to membrane lipids, making them water-insoluble goos that defy purification.
An early attempt to isolate membrane proteins involved what is known as 'acetone
powders'. Tissues are ground up and the aqueous fraction removed by filtration or centrifu-
gation. The non-soluble fraction is then mixed with a large excess of cold acetone that
dissolves the membrane lipids. The solution is then filtered to remove the insoluble proteins.
The filtered protein powder is washed several times with cold acetone and dried producing
the 'acetone powder'. Unfortunately, most proteins comprising the residual protein powder
are denatured and are therefore useless for almost all functional studies. This method of
producing 'acetone powders' is just too harsh for delicate biological membrane material.
However, acetone powers have been successfully employed in some cytoskeletal protein
isolations
[6]
.
A gentler method to isolate membrane proteins is by use of chaotropic agents
[7]
. This
method has also been shown to have unwanted complications, thus limiting its application.
Chaotropic agents act by disrupting inter-molecular interactions including hydrogen
bonding, van der Waals forces, and hydrophobic effects that are responsible at the molecular
level for most biological structure. Chaotropic agents include urea, guanidinium chloride,
lithium perchlorate, Na thiocyanate, NaBr and NaI. When dissolved in water at very high
levels (~4
8 M) chaotropic agents cause membranes to fall apart by eliminating the
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