section 3.3
Separation of Proteins
37
specific activity of the protein. Alternatively, a pure pro-
tein cannot be further subdivided by the methods described
below, e.g., chromatography or electrophoresis.
The initial purification steps usually separate proteins
according to general classification, e.g., fibrous (insolu-
ble) or globular (soluble). The fibrous and globular desig-
nations are related to shape and solubility. Globular pro-
teins are spherical or ellipsoidal and make up the majority
of known proteins. Fibrous proteins contain one or more
polypeptide chains. Their molecules are elongated and
asymmetrical with lengths that may be many times their di-
ameters. Lateral cross-linking between adjacent polypep-
tides, by a variety of types of chemical bonding, confers
mechanical strength and water insolubility to fibrous pro-
teins; consequently, they are found in connective, elastic,
and contractile tissues as well as in hair and skin. An initial
aqueous extraction procedure tends to partition globular
proteins into the soluble fraction and fibrous proteins into
the “insoluble pellet” remaining after centrifugation.
Separation by Molecular Size
Protein can be separated on the basis of their size by
dialysis, gel filtration,
and
membrane filtration.
Small
molecules (e.g., NaCl, amino acids, and sucrose) origi-
nally present or added during the separation of organelles
can be removed by dialysis through a semipermeable
membrane. Dialysis membranes are prepared from cel-
lophane or collodion and contain pores that permit pas-
sage of solute molecules whose molecular weight is less
than ~5000. Thus, proteins of high molecular weight are
retained within a dialysis bag, whereas low-molecular-
weight solutes diffuse through the pores into the fluid
(dialysate) outside of the bag. Complete removal of low-
molecular-weight solutes requires repeated changes of
dialysate. Rapid removal of low-molecular-weight solutes
and simultaneous concentration of the protein solution can
be accomplished by applying pressure to the dialysis solu-
tion (or vacuum to the dialysate). Such a process is known
as ultrafiltration
(Figure 3-1). The principles of membrane
filtration are the same as those of dialysis except that syn-
thetic membranes with specified pore sizes are used.
In gel filtration (or molecular sieving), a column is
packed with hydrated, insoluble gel particles with known
pore sizes; the protein solution is passed through the col-
umn and the effluent solution is collected in fractions that
will contain solutes of different sizes (Figure 3-2). The vol-
ume of the column is essentially divided into two phases:
the gel phase (within the pores) and the solvent phase
(outside the gel particles). As a solution migrates in the
column, the solute molecules that can penetrate the pores
of the gel are distributed both within the gel and outside it.
The particles that are larger than the pore size are excluded
Hydrodynamic
force
— Solvent
• • °0°»-
° %°»o
• C L , » O -
o* o°m
Large,
— impermeable
molecules
Small,
— permeable
molecules
Semi-
sP
membrane
\
I
L
J
o 0
o
o
°o%8o
L°
°
y
FIG U R E 3-1
Separation and concentration of high-molecular-weight solutes from
low-molecular-weight solutes by application of hydrodynamic force over
the solution above a semipermeable membrane (ultrafiltration).
from the gel phase flow through the column more rapidly
and appear first in the effluent. Commonly used gel parti-
cles are inert cross-linked dextrans or agarose, which are
commercially available with a wide range of exclusion
limits. Molecular sieving is effective in the purification of
macromolecules and if the column has been calibrated by
elution of solutes of known molecular weight, it can also
be used in the estimation of molecular weights of proteins
or other solutes.
Separation by Chromatography
Chromatographic separations of proteins are based upon
the differential partitioning of solute molecules due to their
differences in affinity between a moving solvent phase and
a fixed or supportive phase. In gas chromatography, the
mobile phase is a gas, whereas in liquid chromatography
it is a liquid. Gas chromatography is not useful in pro-
tein purification because proteins cannot be converted to
gases without decomposition. Liquid chromatography of
proteins is performed on a variety of mechanically differ-
ent stationary phases, e.g., paper, finely divided particles
coated onto a glass or a plastic surface (thin-layer chro-
matography), or beads packed in a column.
Many chemically different stationary phases are used
in liquid chromatography of proteins.
Ion exchange chro-
matography
uses an ion exchange resin, and the proteins
are eluted with buffer solutions differing in ionic strength
