Biology Reference
In-Depth Information
combining the number of atoms comprising the hydrophilic and hydrophobic portions of the
detergent.
HLB
ΒΌ 20
M
h
=
M
Where M
h
is the molecular mass of the hydrophilic portion of the molecule and M is the
molecular mass of the entire molecule. By this method HLBs can range from 0 to 20 with
0 being a molecule that is completely hydrophobic and 20 a molecule that is completely
hydrophilic. Since HLB is entirely empirical, other formulations can span over different
ranges. A high HLB indicates that the detergent will partition into the aqueous phase while
a low HLB indicates the detergent will prefer remaining in the membrane and so will be very
difficult to extract from the membrane. Detergents used for membrane studies generally have
HLBs between 13 and 15. Structures of several detergents commonly used in membrane
studies are depicted in
Figure 13.1
and their CMCs and HLBs listed in
Table 13.1
. It should
be mentioned that CMCs and HLBs can vary significantly with pH and solvent ionic
strength.
Membrane Detergents
The most suitable detergents for extraction of functional membrane proteins are classified
as non-ionic, zwitterionic or bile salts (detergent structures are shown in
Figure 13.1
).
A neutral detergent that has received a great deal of use in isolating integral membrane
proteins is octyl glucoside (n-octyl-
-D-glucoside). Variations of this basic detergent are
commercially available with mannose replacing glucose and with decyl (10-carbon) or lauryl
(12-carbon) chains replacing octyl (8-carbon). Octyl glucoside is hydrophilic enough to be
readily removed from extracted membrane proteins. CHAPS (3-[(3-cholamidopropyl)dime-
thylammonio]-1-propanesulfonate) is a popular zwitterionic detergent used to solubilize
integral membrane proteins without causing denaturation. Brij-35 (a polyoxyethyleneglycol
lauryl ether) is another non-ionic detergent involved in membrane integral protein extrac-
tions. Brij-35 is also used to prevent nonspecific protein binding to gel filtration and affinity
chromatography supports. A major drawback to Brij-35 is its difficulty in being removed by
dialysis. However, it has been reported that Brij-35 can be removed by Extracti-Gel D Deter-
gent Removing Gel. Triton X-100 is also a non-denaturing, non-ionic detergent. However,
its application in membrane studies is generally different than octyl glucoside or Brij-35.
The major use for Triton X-100 is to lyse or permealize cells. It is not as useful in solubilizing
functional membrane proteins and has difficulty breaking protein
b
protein interactions.
Triton X-100 is also hard to remove from cell membranes. Anyone who has ever tried to
pipette Triton X-100 will remember the unpleasant experience. Triton X-100 is
very
viscous!
Often Triton X-100 is used in conjunction with zwitterionic detergents (such as CHAPS)
that are better suited for membrane protein extractions.
Cholate is the major bile salt comprising
e
80% of all bile salts produced in human liver.
This detergent is a natural product synthesized from cholesterol. Another closely related bile
salt that is popular as a membrane protein extraction detergent is deoxycholate whose struc-
ture differs from cholate only by lacking a single (
w
OH) (
Figure 13.1
). Deoxycholate is
considered to be a secondary bile acid and is a metabolic byproduct of intestinal bacteria.
Both cholate and deoxycholate have been proposed to function in a similar fashion
e
