Download Bioc 462a Lecture Notes

Biochemistry 462a – Proteins: Purification and Characterization
Reading - Chapter 5 and appendix to chapter 5
Practice problems - Proteins extra problems
Protein Purification

Source of Protein
o In order to purify a protein you need a source.
o It might be blood or some other biological fluid, but most often it is a cell, usually
a specific type - liver, muscle, yeast, bacteria, etc.
o The cells must be broken open - homogenized - to release the protein in a soluble
form.
o Homogenization conditions must be worked out that release the protein from the
cell without damaging the protein.
o Membrane-bound proteins can also be purified, but different approaches are
required.

Fractional Precipitation
o In concentrated salt solutions, usually ammonium sulfate is used; some proteins are
more soluble than others. By varying the concentration of ammonium sulfate, one
can achieve some limited purification of proteins. This technique is often used in the
first step of protein purification. In general,
o Small proteins are more soluble than large proteins.
o The larger the number of charged side chains, the more soluble the protein.

Column Chromatography
o The invention of column chromatography was a critical event in biochemistry,
because it was the basis for the development of procedures for obtaining pure
proteins.
o Studies on pure proteins are essential for understanding the structural and functional
properties of proteins.
o In column chromatography an absorbent (see below) is placed in a glass tube.
o A protein mixture is passed into the column and binds to the adsorbent.
o By proper choice of the eluting buffer, specific proteins can be eluted from the
absorbent and separated from other proteins in the mixture.
o By repeating this procedure with several different absorbents, pure protein can be
obtained.
o Because proteins are not very stable, low temperature (4o C) and neutral pH must
often be employed.
o The properties of some adsorbents are described below.

Ion Exchange Chromatography
o Ion exchange resins have fixed charges - either positive or negative.
o Proteins bind to the resin via electrostatic interactions.
o The strength of these interactions depends on the net charge on the protein, which is a
function of pH and the nature of the weak acid amino acid side chains, and the salt
concentration of the buffer - high salt concentrations reduce the interaction.

Affinity Chromatography
o This is a more specific interaction in which a ligand specifically recognized by the
protein of interest is attached to the column material.
o When a mixture of proteins is passed through the column, only those few that bind
strongly to the ligand will stick, while the others will pass through the column.
o By changing the buffer one weakens the interaction between the protein and the
ligand, which causes the protein to be eluted from the column.
o A variation is immunoaffinity chromatography; in which an antibody specific for a
protein is immobilized on the column and used to affinity purify the specific protein.

Gel Filtration Chromatography
o The column consists of material that separates proteins based on their size and shape.
o A wide range of molecular exclusion limits is available for separating proteins of all
sizes.
o For any particular column dimensions and
material, the volume of buffer required to elute a
specific protein depends on the molecular weight
of the protein. Thus, one can separate proteins by
size.
o If one calibrates the column by determining the
elution volume of proteins with known molecular
weights, then a calibration curve relating elution volume and molecular weight can be
constructed.
o
Such a calibration curve can then be used to estimate the molecular weight of an
unknown protein.

Dialysis/Ultrafiltration
o Semipermeable membranes are available, which allow
passage of small molecules but exclude the passage of
proteins. Sacs made of such material allow the salt
and buffer components of a protein solution to be
changed to another buffer

Practical Example







This figure illustrates several of the techniques discussed above. It is taken from
"Isolation, Characterization, and cDNA Sequence of Two Fatty Acid-Binding Proteins
from the Midgut of Manduca sexta Larvae". A. F. Smith, K. Tsuchida, E. Hanneman, T.
C., Suzuki, and M. A. Wells, J. Biol. Chem. 267, 380-384 (1992).
This is the elution profile from an anion exchange resin (binds negatively charged
proteins). The proteins were eluted by increasing the NaCl concentration in the eluting
buffer.
Total protein was measured by determining the absorbance at 280 nm.
In order to follow the fatty acid-binding proteins, they were labeled by binding
radioactive fatty acids (CPM=counts per minute - gray shading).
The purity of each peak was assessed using SDS-PAGE (insert).
There are two, nearly pure, proteins that bind fatty acids.
The two proteins were obtained in pure form following one additional step.
Protein Characterization





Electrophoresis
o In an electric field a protein or other charged
macromolecule will move with a velocity that
depends directly on the charge on the
macromolecule and inversely on its size and shape.
Gel electrophoresis is carried out in some supporting
media, usually polyacrylamide or agarose, which has
pores of sufficient size to allow passage of the
macromolecule.
o The proteins in the gel are easily stained for
detection purposes.
o Because the net charge on a protein and its molecular weight are characteristic
properties of a protein, electrophoresis is a powerful method for characterizing the
purity of a protein preparation.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a variant of
electrophoresis in which the buffers contain SDS, a detergent that binds to proteins.
o Most proteins bind SDS at a constant ratio, about 1 SDS for every 2 amino acids.
o The large negative charge resulting from the bound SDS masks the native charge on
the protein, so that all proteins have essentially the same charge to mass ratio.
o This means that that the rate of movement in the electric field depends only on the
molecular weight.
o In addition, the SDS causes all proteins to adopt a random-coil structure, which
means that shape does not effect movement through the gel.
o Thus SDS-PAGE is very useful method for determining the molecular weight of a
protein.
Western blotting is a technique for detecting a specific protein in a mixture.
Gel electrophoresis and SDS-PAGE electrophoresis are primarily useful as analytical
techniques, although they can be used for purification.
o In this figure, lane 1 would contain standards of
known molecular weight, lane 2 a mixture of three
unknown proteins and lanes 3-5 the three unknown
proteins.
o SDS-PAGE can be used to determine the molecular
weight of a protein.
o The molecular weights of the three unknown proteins
can be determined from a calibration curve
constructed by plotting the log of the molecular
weight of the standard proteins vs. the distance
traveled in the gel.
o The distance traveled depends on the porosity of the
gel. The lower the porosity the further the proteins
move.
o


Depending on the molecular of the unknown proteins, different porosity gels will
need to be used.
Isoelectric Focusing
o In this technique electrophoresis
occurs through a stable pH gradient.
o Under these conditions the proteins
will migrate in the electric field until
they reach a point in the pH gradient
where their net charge becomes 0 - the
isoelectric point.
o The isoelectric point depends on the
exact number and type of weak acid
amino acid side chains present in the
protein. Therefore, isoelectric focusing
is a useful purification procedure.
o Often isoelectric focusing is combined
with SDS PAGE in two-dimensional electrophoresis.
Molecular Weight and Shape are fundamental physical properties of a protein.
o Estimates of molecular weight can be obtained using SDS-PAGE or gel filtration, as
described above.
o One very useful technique for measuring molecular weight and shape is
centrifugation.
 A particle that is subjected to a centrifugal field by being spun in a centrifuge
is subjected to a force,
, where m is the mass of the mass of
the particle, r is the distance of the particle from the center of rotation, and
is the angular velocity.

o
o
o
is the buoyancy factor which accounts for the fact that particle is
buoyed up by the surrounding solvent of density (g/ml).

is the specific volume of the particle (ml/g) (= 1/density of the particle).
 If = then the particle will not move.
The movement of the particle through the solvent is resisted by a frictional
coefficient, f, that depends on the shape of the particle.
 The frictional coefficient is an important factor in any transport process, such
as centrifugation or gel filtration.
 A spherical particle has a f = 1.0, whereas a cigar-shaped or cylindricallyshaped particle will have f > 1.0.
The movement of any particle under the influence of a centrifugal field is
characterized by its sedimentation coefficient, S, which is directly proportional to its
molecular mass, M, and inversely proportional to f.
, where N is Avogadro's number.

Ultracentrifugation is used in two ways to characterize proteins
In sedimentation equilibrium
experiments, the centrifuge is operated at
a relative low speed so that the forces of
sedimentation and diffusion balance and
the protein distributes in the centrifuge
cell in a manner proportional to its
molecular weight.
In sedimentation velocity experiments,
the centrifuge is operated at maximal
speed, which causes the protein to
sediment to the bottom of the tube. The
rate at which the boundary moves gives
S, which when combined with M gives f,
a measure of the shape of the protein.
Three Dimensional Structure



Whenever possible, it is highly desirable to obtain the three dimensional structure of a
protein.
Most often, this is done by X-ray crystallography, although NMR can be used, especially
with small proteins.
It is impressive to note that more than 10,000 structures have been determined, most in
the last decade, as new, more powerful instruments have become available.
Structural Homology

In addition to sequence homology for proteins with identical functions from different
organisms, there are often domains in a protein that are conserved. For example, most
proteins that bind nucleotides, such as ADP, have a common nucleotide-binding motif.

There are even a few cases in which proteins with entirely different functions have very
similar three-dimensional structures, as shown below for lysozyme, an enzyme, and lactalbumin, a milk protein.
Lysozyme
Lactalbumin