Asymmetry of apolipoprotein A-I in solution as assessed from ultracentrifugal, viscometric, and fluorescence polarization studies

Abstract

The solution properties of apoliporprotein A-I (apo A-I) isolated from human and rhesus monkey serum highdensity lipoproteins were examined by the techniques of fluorescence polarization, sedimentaion velocity, sedimentation equilibrium, and viscometry as a function of protein concentration. The ultracentrifugal studies confirmed previous reports that human and rhesus monkey apo A-I undergo a concentration-dependent, reversible self-association in aqueous solutions. The sedimentaion velocity data, extrapolated to infinite dilution, indicated that the apo A-I monomer is asymmetric, with an axial ratio of about 6. The reduced viscosity of apo A-I solutions was also markedly affected by protein concentration; within the range of concentrations studied, the values for human apo A-I ranged from 11.19 to 21.24 mL/g and for rhesus apo A-I, from 8.26 to 16.83 mL/g. The intrinsic viscosity, 6.58 mL/g, of the apo A-I monomer was significantly higher than that of a typical globular protein (3-4 mL/g); the axial ratio, 5.5, was in agreement with that obtained from the ultracentrifugation analyses. In the fluorescence polarization studies, human and rhesus apo A-I were dansylated to the extent of 1.0 to 2.5 mol of dansyl group/mol of apo A-I. This chemical modification was attended by no appreciable change in the secondary structure of apomore » A-I, as assessed by circular dichroism spectra. The apparent sedimentation coefficient, molecular weight, and rotational relaxation time of each of the dansylated proteins indicated that both human and rhesus monkey apo A-I self-associated to a lesser extent than the unmodified apo A-I. At protein concentrations ranging from 0.002 to 3.0 g/L, the fluorescence polarization values of dansylated apo A-I remained essentially constant (0.185 to 0.192), although rotational relaxation times indicated self-association at the higher concentrations.« less