Sodium dodecyl sulfate-polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin.
Avidin, a positively charged egg-white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS-PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS-PAGE.
Bayer
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EA
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel. bfbayer@wiccmail.weizmann.ac.il
Ehrlich-Rogozinski
S
S
Wilchek
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M
D001360:Avidin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006358:Hot Temperature; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D019809:Streptavidin
D001426:Bacterial Proteins; D001360:Avidin; D012967:Sodium Dodecyl Sulfate; D019809:Streptavidin