Human Biochemistry Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Proteins: Structure and Properties
Determination of Quaternary Structure

Determining The quaternary Structure of Oligomeric Proteins involves identifying the protomers of each type, establishing their relative orientation, and investigating the interactions that stabilize the overall structure.

Many Methods available for determining the molecular mass of an oligomer are suitable as long as they do not cause the oligomer to denature. However, if the oligomer is denatured beforehand, the molecular mass of its protomers can be determined using the exact same methods.

Ultracentrifugation

This method, developed by Svedberg, is based on measuring The rate of sedimentation (precipitation) under centrifugal forces generated in an ultracentrifuge at accelerations on the order of 10⁵g.

Sucrose density gradient centrifugation

A set of protein standards and the protein under investigation are layered on top of a sucrose solution poured into a plastic centrifuge tube. The density of this solution varies to form a concentration gradient from 5 to 20%; centrifugation is then carried out overnight at ~10⁵g. Afterward, the bottom of the tube is punctured with a needle, the contents are collected drop by drop into small tubes, the relative positions of the proteins along the density gradient are determined, and the necessary calculations are performed.

Molecular sieve filtration

First, a Column packed with Sephadex or a similar matrix is calibrated using a set of proteins with known molecular weights. The molecular mass of an unknown protein is then estimated by comparing its mobility with that of the standards. However, if the protein is a highly asymmetric molecule or interacts with the matrix (molecular sieve), significant errors may arise.

Class="center">Table 5.6. Quaternary Structure of selected Enzymes

¹⁾ From the review by Klotz I. M., Langerman N. R., Darnall D. W.: Quaternary METABOLISM/9.html">Structure of enzymes. Annu. Rev. Biochem. 1970;39:25.

²⁾ Non-identical subunits.

Polyacrylamide gel Electrophoresis

A set of standard proteins is separated by electrophoresis in gels of varying porosity with a cross-linking degree of 5–15%. Proteins are visualized by staining with Coomassie Brilliant Blue or silver, and their molecular mass is determined by comparing their mobility with that of the standards. This method has found particularly wide application in determining the molecular mass of protomers; the oligomer is first denatured (e.g., by boiling in a detergent in the presence of ß-mercaptoethanol) and then separated in gels containing an ionic detergent, sodium dodecyl sulfate.

Electron Cell/15.html">Microscopy

Electron microscopes make it possible to obtain images of small objects at linear magnifications of up to 100,000 times. This opens up the possibility of visualizing high-molecular-weight proteins within Viral Particles, enzyme complexes, and oligomeric proteins.

Table 5.6 presents selected data on the number and molecular mass of protomers that form the quaternary structure of various enzymes.

References

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