Dye-labelling as a means to study ternary protein complexes by analytical ultracentrifugation: the band 3/ankyrin/aldolase complex from erythrocyte membranes
Demonstration of the formation of ternary complexes of proteins, in the presence of all constituents and binary complexes, and analysis of their stoichiometries is a difficult task. For the band 3/ankyrin/aldolase complex from erythrocyte membranes in detergent solutions, we have solved this problem by sedimentation equilibrium analysis in the analytical ultracentrifuge. Labelling of the ankyrin with a dye (fluorescein isothiocyanate) and measuring the absorbance versus radius profiles at a wavelength where only the dye absorbs allowed us to focus on the ankyrin-containing complexes. So, the different oligomers of uncomplexed band 3, the uncomplexed aldolase and the various binary complexes of band 3 and aldolase could be disregarded in the analysis. The ternary band 3/ankyrin/aldolase complex could be unambiguously detected. Its stoichiometry (band 3/ankyrin/aldolase tetramer) was found to vary between 4:1:1 and 4:1:4, depending on the abundancy of the enzyme.
Key wordsSedimentation equilibrium ternary protein complexes dye labelling band 3/ankyrin/aldolase complex
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