Quaternary structure of apolipoprotein E in solution: fluorimetric, chromatographic and immunochemical studies.
Self-association and stability in solution of apolipoprotein E (apoE), isolated from human plasma very low density lipoproteins, were studied in the nanomolar concentration range. Equilibrium denaturation of fluorescein-labelled apoE (induced by guanidine hydrochloride) was studied by measurement of fluorescence anisotropy, total fluorescence emission intensity, the shift in wavelength of maximal fluorescence emission, and gel-chromatographic behaviour. The protein denaturation was reversible, displayed biphasic behaviour, and was dependent on the apoE concentration. As measured by fluorescence anisotropy, the kinetics of apoE denaturation in the presence of 6M denaturant were heterogeneous, and the contribution of the long-lived component increased with the apoprotein concentration. The results are in agreement with the following scheme: Oligomer (in aged preparations) in equilibrium with tetramer in equilibrium with native or partially denatured monomer in equilibrium with fully denatured monomer. It is suggested that self-association of individual apoE molecules in solution is due to their lipid-binding domains, and leads to additional stabilization of apoprotein structure. Monoclonal antibody 3D12F11 of the IgG1 subclass bound with high affinity to apoE (Kd = 3.5 +/- 0.5 nM), and had no effect on apoprotein binding to heparin-Sepharose or the apoprotein-induced destabilization of liposomes formed from dipalmitoyl-phosphatidylcholine. This indicates that the epitope to the antibody is localized outside the heparin- and lipid-binding sites of the apoprotein molecule.
Dergunov
A D
AD
Department of Biochemistry, USSR Research Centre for Preventive Medicine, Moscow.
Shuvaev
V V
VV
Yanushevskaja
E V
EV
D000911:Antibodies, Monoclonal / Q000276:immunology; D001057:Apolipoproteins E / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D001665:Binding Sites; D002850:Chromatography, Gel; D000939:Epitopes / Q000032:analysis; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; D000939:Epitopes; D006146:Guanidines; D046911:Macromolecular Substances; D019791:Guanidine