Some negative staining electron microscopic and biochemical studies on apoferritin and its oligomers

Abstract

Native horse spleen apoferritin, which is a mixture containing predominantly the 17S monomer with gradually decreasing amounts of dimer, trimer, tetramer, pentamer and higher oligomers, has been studied by polyacrylamide gel electrophoresis in non-dissociating systems, analytical ultracentrifugation and negative staining electron microscopy. The apoferritin monomer has been purified by preparative polyacrylamide gel electrophoresis and gel slicing of analytical polyacrylamide gels. From an oligomer enriched fraction obtained by Sepharose 6B gel filtration chromatography, the dimer and trimer have been successfully purified and a tetramer + higher oligomer fraction obtained by gel slicing of analytical polyacrylamide gels. The stability of the separated fractions during electrophoresis and negative staining is clearly demonstrated. The oligomer fractions can, however, be readily dissociated into monomer and lower oligomers by subjecting them to ultrasonication, the degree of dissociation being time dependent at a fixed ultrasonication intensity. The number of possible conformations for the higher oligomers clearly increases with the number of monomers present in the group, resulting in varied electron optical images of the apparently randomly linked monomers. The production of an increased amount of inter-molecular cross-linking has been achieved by treatment with low concentrations of glutaraldehyde (up to 0.05%). This treatment produced a diminution of the monomer band on polyacrylamide electropherograms, with an increased amount of higher oligomers. Oligomers above the pentamer are not clearly revealed by the electrophoretic systems used, owing to their inability to resolve the varied molecular groupings as discrete stained bands. The electron microscope has the ability to reveal whether or not the higher oligomers contain compact groups or extended chains of molecules.