3.5

The three
most widely used characteristics for separating proteins are
, defined as either length or mass; ;
and for specific ligands.

The first step in a typical protein purification scheme is

Proteins vary
greatly in mass, but not in .

The , s, of a protein
is a measure of its sedimentation rate. The sedimentation
constant is commonly expressed in units (S),
where a typical large protein complex is about 3–5S, a proteasome
is 26S, and a eukaryotic ribosome is 80S.

The most common initial step
in protein purification from cells or tissues is the separation
of water-soluble proteins from insoluble cellular material by
. A starting mixture, commonly a
cell homogenate (mechanically broken cells), is poured into a
tube and spun at a rotor speed, and for a period of time, that
forces cell organelles such as nuclei as well as large unbroken
cells or large cell fragments to collect as a pellet at the bottom;
the soluble proteins remain in the supernatant

On the basis of differences in
their masses, water-soluble proteins can be separated by
centrifugation through a solution of increasing density,
called a .

All
the proteins start from the thin layer of the sample that was
placed at the top of the tube and separate into bands (actually,
disks) of proteins of different masses as they travel at
different rates through the density gradient. In this separation technique,
called , samples are centrifuged
just long enough to separate the molecules of interest
into discrete bands, also called zones.

In - gel electrophoresis, proteins are separated
sequentially, first by their and then by their

In two-dimensional gel electrophoresis, proteins are separated
sequentially, first by their charges and then by their
masses (Figure 3-39a). In the first step, a cell or tissue extract
is fully denatured by high concentrations (8 M) of urea
(and sometimes SDS) and then layered on a strip of gel that
contains urea, which removes any bound SDS, and a continuous
pH gradient. The pH gradient is formed by ampholytes,
polyanionic and polycationic small molecules that are
cast into the gel. When an electric field is applied to the gel,
the ampholytes will migrate. Ampholytes with an excess of
negative charges will migrate toward the anode, where they
establish an acidic pH (many protons), while ampholytes
with an excess of positive charges will migrate toward the
cathode, where they establish an alkaline pH. The careful
choice of the mixture of ampholytes and careful preparation
of the gel allows the construction of stable pH gradients
ranging from pH 3 to pH 10. A charged protein placed at
one end of such a gel will migrate through the gradient under
the influence of the electric field until it reaches its (pI), the pH at which the net charge of the protein is
. With no net charge, the protein will migrate no further.
This technique, called (IEF), can resolve
proteins that differ by only one charge unit. This method is
sensitive enough to separate phosphorylated and nonphosphorylated
versions of the same protein.

Proteins that have been separated on an IEF gel can
then be separated in a second dimension on the basis of
their molecular weights. To accomplish this separation, the
IEF gel strip is placed lengthwise on one outside edge of a
square or rectangular slab of polyacrylamide gel, this time
saturated with SDS to confer on each separated protein a
more or less constant : ratio.

In this technique, called (LC),
the sample is placed on top of a tightly packed column of
spherical beads held within a glass, metal, or plastic cylinder
( Figure 3-40). The sample then flows down the column,
driven by gravitational or hydrostatic forces alone or sometimes
with the assistance of a pump.

Proteins that differ in mass
can be separated on a column of porous beads made from
polyacrylamide, dextran (a bacterial polysaccharide), or agarose
(a seaweed derivative)—a technique called

Although proteins flow around the beads,
they spend some time within the large depressions that cover
a bead’s surface. Because smaller proteins can penetrate these
depressions more readily than larger proteins can, they travel
through a gel filtration column more than larger
proteins do (Figure 3-40a). (In contrast, proteins migrate
through the pores in an gel; thus smaller
proteins move than larger ones.)

The purification of a protein, or any other molecule, requires
a specific that can detect the presence of that molecule
as it is separated from other molecules (e.g., in column or
density-gradient fractions or gel bands or spots).

Many assays are tailored
to detect some functional aspect of a protein. For example,
assays of enzymatic activity are based on the ability to detect
the loss of substrate or the formation of product, these are called

The classic method for determining the amino acid sequence
of a protein is

In this procedure (Edman degredation), the
free of the N-terminal amino acid of a polypeptide
is labeled, and the labeled amino acid is then cleaved
from the polypeptide and identified by . The polypeptide is left one residue
shorter, with a new amino acid at the N-terminus. The cycle
is repeated on the ever-shortening polypeptide until all the
residues have been identified.

A is
the list of the molecular weights of peptides that are generated
from the protein by digestion with a specific protease,
such as trypsin.

In regards to X-Rar Crystallography: Elaborate
calculations and modifications of the protein (such as the
binding of heavy metals) must be made to interpret the diffraction
pattern and calculate the distribution of electrons
(called the ).

In this
technique, a dilute protein sample in an aqueous solution
is applied in a thin layer to an electron microscope sample
holder (a “grid”) and rapidly frozen in liquid helium to preserve
its structure. It is then examined in the frozen, hydrated
state in a microscope.

An important distinction between x-ray crystallography
and NMR spectroscopy is that the former method directly
determines the of the atoms, while the latter
directly determines the between the atoms, from
which the structure is deduced.

Centrifugation separates proteins on the basis of their
rates of , which are influenced by their masses
and shapes

separates proteins on the basis of
their rates of movement in an applied electric field. SDSpolyacrylamide
gel electrophoresis (SDS-PAGE) can resolve
polypeptide chains differing in molecular weight by
10 percent or less (see Figure 3-38). Two-dimensional gel
electrophoresis provides additional resolution by separating
proteins first by (first dimension) and then by
(second dimension).

separates proteins on the basis
of their rates of movement through a column packed with
spherical beads. Proteins differing in mass are resolved on
gel filtration columns; those differing in charge, on ionexchange
columns; and those differing in ligand-binding
properties, on affinity columns (see Figure 3-40).

, often abbreviated as IP, permits the
separation of a protein of interest from other proteins in a
complex mixture using antibodies specific for the protein of
interest. The antibodies are used to precipitate their target
protein out of solution for subsequent analysis. Molecules
tightly bound to the target protein can precipitate with it

- experiments can determine the intracellular
fate of proteins and other metabolites

is a technique for detecting radioactively
labeled molecules in cells, tissues, or electrophoretic gels using
two-dimensional detectors (photographic emulsion or
electronic detectors).

provides the most detailed
structures but requires protein .
microscopy is most useful for large protein complexes,
which are difficult to crystallize. Only relatively small proteins
are amenable to three-dimensional structural analysis.