Apolipoprotein E self-association in solution studied by non-radiative energy transfer.
The self-association of human apolipoprotein E (apoE), isolated from plasma very low density lipoproteins, was studied at apoE concentrations less than 0.6 microM by non-radiative energy transfer. ApoE was separately labeled with a fluorescent donor group i.e. dansyl chloride (apoE/D) and with an acceptor i.e. fluorescein isothiocyanate (apoE/F). Mixed apoE/D:apoE/F complexes were prepared either by incubation or the donor- and of the acceptor-labeled apoE or by renaturation during dialysis of the apoE/D:apoE/F mixture pre-denatured by addition of guanidine hydrochloride or by treatment with sodium cholate. The efficiency of energy transfer E at an equimolar ratio of the donor to acceptor and a ratio of 1.9 mol fluorescein/mol protein amounted to 29.2 +/- 2.6% (n = 3). The E value increased linearly with increasing acceptor fraction in the mixture. The state of self-association of apoE as tetramers within this concentration range was confirmed by cross-linking experiments with a water-soluble bifunctional reagent. This approach can be applied to the study of protein-protein interactions in apolipoprotein-phospholipid recombinants.
Dergunov
A D
AD
Department of Biochemistry, Research Centre for Preventive Medicine, Moscow, Russian Federation.
Vorotnikova
Y Y
YY
De Pauw
M
M
Rosseneu
M
M
D001057:Apolipoproteins E / Q000097:blood / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003619:Dansyl Compounds; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007700:Kinetics; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D001057:Apolipoproteins E; D003619:Dansyl Compounds; D012996:Solutions; D016650:Fluorescein-5-isothiocyanate; C029038:dansyl chloride