{*Figure 11a*}Protein/Enzyme Purification
Section E
Separation of Proteins by Biological Activity or Function
Biological Activity and Function depend on binding of substrate (metabolite) to enzyme (protein):
{*Figure 11a*}
{*Figure 11b*}
Illustration of glucose (small dark blue molecule) binding to hexokinase (an enzyme).
In this case, the enzyme acts like a jaw and clamps down on the substrate (glucose).
[This figure (11a & 11b) is from Voet & Voet Biochemistry Text ©1990 John Wiley & Sons]
Affinity chromatography depends on the biological activity or the affinity of the enzyme for its substrates.
It is usually a very effective method of purification.
Sometimes, a protein can be purified to homogeneity (complete purity) using only affinity chromatography.
Concept of Affinity chromatography:
{*Figure 12a*}
First, the " affinity material " is made by attaching a substrate (or inhibitor) to an inert support like a Gel bead by covalent chemistry, sometimes a spacer arm is used to make substrate more accessible to the enzyme. Next, a column is prepared with affinity material and a mixture of proteins is applied to the column.
Specific Binding in Affinity Chromatography
{*Figure 12b*}
Specific enzyme (special shape in figure) binds to the covalently attached substrate on the affinity material. Other proteins with no affinity for substrate are washed away by buffer.
Elution of Specifically Bound Enzyme:
{*Figure 12c*}
Enzyme is eluted by applying free substrate in buffer. Enzyme has higher affinity for free substrate than bound substrate, so it is eluted from affinity material.
Finally, the free substrate can be removed from enzyme by dialysis or gel filtration.
[This figure (12a, 12b & 12c) is from an unknown source who owns the copyright]
SUMMARY of PURIFICATION PROCEDURES
To obtain a homogeneous protein/enzyme, one must apply a combination of the methods.
For example, ion-exchange chromatography is applied first, which separates proteins by difference in ionic properties;
Then gel filtration is applied, which separates by difference in molecular size.
Since few proteins have exactly the same ionic character and same molecular size,
a high degree of purity will be achieved by a combination of these two purification methods.
Affinity chromatography, which is much more specific for a particular enzyme, will in the end usually render an enzyme homogeneous.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu