PHYSICAL-PROPERTIES OF APOLIPOPROTEIN-A-I FROM THE CHICKEN, GALLUS-DOMESTICUS

The amphipathic alpha-helices of exchangeable apolipoproteins (apo) fu nction to simultaneously facilitate interaction with lipid surfaces an d the aqueous environment. In contrast to mammalian apoA-I's, which se lf-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein wh en dissociated from high-density lipoprotein (HDL). Sedimentation equi librium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28 170. Corresponding sedimentation velocity and diffusion experiments gave rise to s0(20,w) = 2.23 S and D0(20,w) = 6.39 X 10(-7) cm2/s. A translational frictional ratio (f/f (min)) of 1.18 and an axial ratio of 4.0 were also determined from thi s data. The Stokes radius (R(s,sed) = 2.80 nm) and translational frict ional ratio were subsequently used to calculate estimated molecular di mensions of 25.2 x 100.8 angstrom for chicken apoA-I. Circular dichroi sm (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glockner analysis. Denaturation studies performe d on lipid-free apoA-I and monitored by CD revealed a midpoint of dena turation of 0.64 M guanidine hydrochloride. From plots of DELTAG(app) versus guanidine hydrochloride concentration, a DELTAG(D)H2O of 1.86 k cal/mol was determined. In other studies, a midpoint of temperature-in duced denaturation for apoA-I of 57-degrees-C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggest ing that lipid-free apoA-I may by partially stabilized by the formatio n of intra- or interhelix salt bridges between oppositely charged amin o acid side chains. Denaturation studies of apoA-I bound to the surfac e of HDL revealed a midpoint of guanidine hydrochloride induced denatu ration of 3.25 M, indicating that considerable stability is conferred on this protein through interaction with lipid. The relative lipid bin ding affinities of chicken and human apoA-I's were examined in displac ement studies employing a model lipid surface, insect low-density lipo phorin. The data showed that chicken apoA-I has a slightly higher affi nity for lipid surfaces than its human counterpart. The results are di scussed in relation to the known structural properties of human apoA-I .