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 s odium dodecyl sulfate (SDS). The resultant aggregates fail to penetrat e the stacking gel during polyacrylamide gel electrophoresis (PAGE). T o prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a ma nner similar to that of streptavidin; under nondenaturing conditions ( i.e., incubation of samples at room temperature), both proteins normal ly migrated mainly as tetramers with a tendency to form oligomers of t he tetramer. When samples were boiled, both proteins migrated mainly a s the monomer. The comparative stability properties of avidin and stre ptavidin were also examined using SDS-PAGE by heating samples and dete rmining the extent of dissociation of tetramers to monomers as a funct ion of temperature. A distinctive transition temperature could be defi ned 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 structure s of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structur e of the molecule using SDS-PAGE.