40
chapter 3
Protein Isolation and Determination of Amino Acid Sequence
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Capture column
(glutathione bound to agarose)
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Thrombin column
(cleavage of GST and protein)
Separation and
purification column
GST
Protein
FIG U R E 3-4
A schematic diagram of the steps in the purification of a protein fused with glutathione S-transferase. Protein
purification using this procedure utilizes three columns and is fully automatic.
charged protein molecules migrate toward either the anode
or the cathode. Factors that influence the rate of migration
are pH, composition of the medium through which migra-
tion occurs, and size and shape of the protein molecule.
A protein that does not migrate in an electric field at a
given pH has no net charge at that pH. That pH is called
the
isoelectric point
(pi) of the protein. The pi value is
characteristic for each protein (Table 3-1). In a solution
at a pH value above its pi, a protein will have a net nega-
tive charge; below its pi, a protein will have a net positive
charge. The electrophoresis technique known as
isoelec-
tric focusing
separates proteins on the basis of differences
in their isoelectric points. Proteins whose pi values dif-
fer by as little as 0.02 pH unit can be separated by this
technique. In isoelectric focusing, a mixture of proteins is
placed in a pH gradient; in the presence of an electric field,
each protein migrates to a position corresponding to its pi
and comes to rest in a narrow band at that pH. The pH gra-
dient is established by placing in an electric field an aque-
ous mixture of synthetic low-molecular-weight (300-600)
ampholytes (oligomers of aliphatic amines, amino acids,
and carboxylic acids). On application of an electric poten-
tial, the ampholytes migrate and come to rest according
to their respective isoelectric points. If proteins are mixed
with the ampholytes, the proteins migrate to the positions
of their respective isoelectric points within the ampholyte
gradient and can thereby be separated and concentrated
into narrow bands (Figure 3-5).
Electrophoretic techniques also yield estimates of the
molecular weights of proteins and nucleic acids. The de-
tergent sodium dodecyl sulfate (SDS) and proteins form
SDS-protein complexes that migrate in polyacrylamide
gels according to their molecular weights. SDS dissociates
multi subunit proteins into individual polypeptide chains.
Each denatured chain has a uniform negative charge per
unit mass of protein, since the total negative charge of
the sulfonic acid groups of SDS, which are uniformly
located along the surface of the protein, far exceeds the
T A B LE 3-1
Isoelectric Points of Some Proteins
Protein
Pi
Lysozyme
1 1 . 0
Cytochrome C
1 0 . 6
Pancreatic ribonucléase
9.6
Chymotrypsinogen
9.5
Myoglobin
7.0
Human growth hormone (somatotropin)
6.9
Hemoglobin
6 . 8
Human serum immunoglobulins
6.4-7.2
Carboxypeptidase
6 . 0
Catalase
5.6
Fibrinogen
5.5
/TLactoglobulin
5.2
Urease
5.1
Human serum albumin
4.8
Egg albumin
4.6
Thyroglobulin
4.6
Pepsin
1 . 0
