{*Figure 19*}Protein/Enzyme Purification -- Part II
Section D
Determining the Number of Subunits by SDS-PAGE
An easy way to analyze the subunit composition of enzyme is...
denature the protein in the presence of detergent Sodium DodecylSulfate (SDS) and...
run a polyacrylamide gel on the denatured polypeptide.
This is called SDS-PAGE or Denaturing PAGE.
Illustration of Protein Treatment with SDS and Heat for Denaturation
{*Figure 19*}
Native protein is unfolded by heating in the presence of a disulfide bond reductant and SDS.
Many proteins contain disulfide bonds (Cys-S-S-Cys) joining the polypeptide backbone and...
to remove these bonds, a disulfide reducing agent (like beta-mercaptoethanol) is used.
Disulfide reducing agents convert disulfide bonds (Cys-S-S-Cys) to thiols (Cys-SH).
During heating the protein would normally precipitate,
but the SDS binds to the backbone and provides a negative charge to make the denatured protein soluble.
SDS, like all detergents, has a hydrophobic tail and a charged polar ion.
The hydrophobic tail (ie the dodecyl part of SDS) binds to the hydrophobic backbone of the protein,
and the ionic sulfate group projects out into solution making the denatured protein soluble.
Large polypeptides bind more SDS than small polypeptides.
So proteins end up with negative charge in relation to their size.
During electrophoresis, large proteins move less distance in the gel than small proteins.
This is because the protein/SDS ratio is constant for all proteins and...
the gel structure controls protein movement by friction.
So bigger polypeptides move slower during the electrophoresis.
Overall, this results in electrophoretic mobility for polypeptides
in the SDS PAGE gel in relation to subunit molecular weight (MW).
The MW of Protein Subunits Estimated by SDS-PAGE
Separate denatured polypeptides by PAGE, which is usually called SDS-PAGE.
Estimate MW of subunit by comparison to polypeptides of known MW,
which have also been denatured in presence of SDS.
Illustration of an SDS-PAGE gel:
{*Figure 20*}
The SDS-PAGE gel illustrated here is for a complete purification of an enzyme. The protein mixtures obtained at each step in the purification are run in a lane of a slab gel (see Fig. 13 above).
The Purification steps and the lane labels on the gel are:
1. Crude Extract -- total mixture of all proteins at the start of the purification.
2. After Ion Exchange Chromatography -- containing enzyme activity of interest and a mixture of proteins.
3. After Gel Filtration Chromatography -- containing enzyme activity of interest and a mixture of proteins.
4. After Affinity Chromatography -- containing enzyme activity of interest and a single protein.
5. Standard Proteins of known molecular weight for their subunits.
The SDS-PAGE gel can only be stained for protein since the proteins are denatured and...
no longer have any biological activity.
Calibration Plot for an SDS-PAGE Gel
{*Figure 21*}
The log of the molecular weight (MW) is plotted versus the electrophoretic mobility of the standard proteins.
(Electrophoretic mobility means how far the protein moved in the gel during electrophoresis).
The standard proteins have known subunit molecular weights.
Using the plot, the MW of the unknown pure protein is determined.
Only pure proteins can be used for estimating subunit MW,
since the presence of contaminating proteins would lead to confusion.
Determining the Subunit Composition with Native MW and Subunit MW
By combining the information on the native MW,
the subunit MW and the number of different polypeptide chains -
you can figure out what the subunit composition of the enzyme is.
For example, by gel filtration Native MW = 49,000.
One subunit polypeptide was found with MW = 25,000.
Therefore, the protein/enzyme exists in solution as a dimer (ie 49,000/25,000 = 2).
In other words, divide the Native MW by the Subunit MW to find an integer.
Summary
Two types of polyacrylamide gel electrophoresis are used in protein purification/characterization.
1. Native PAGE to test for purity.
2. Denaturing or SDS-PAGE to determine subunit MW.
One can also determine by SDS-PAGE if the protein contains subunits of different size.
For example, a protein may contain two kinds of subunits and these may have a different size.
Some enzymes have a catalytic subunit and a regulatory subunit.
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu