Surface rheology and adsorption kinetics reveal the relative amphiphilicity, interfacial activity, and stability of human exchangeable apolipoproteins

Abstract

Exchangeable apolipoproteins are located in the surface of lipoprotein particles and regulate lipid metabolism through direct protein-protein and protein-lipid interactions. These proteins are characterized by the presence of tandem repeats of amphiphatic alpha-helix segments and a high surface activity in monolayers and lipoprotein surfaces. A noteworthy aspect in the description of the function of exchangeable apolipoproteins is the requirement of a quantitative account of the relation between their physicochemical and structural characteristics and changes in the mesoscopic system parameters such as the maximum surface pressure and relative stability at interfaces. To comply with this demand, we set out to establish the relations among alpha-helix amphiphilicity, surface concentration, and surface rheology of apolipoproteins ApoA-I, ApoA-II, ApoC-I, ApoC-II, and ApoC-III adsorbed at the air-water interface. Our studies render further insights into the interfacial properties of exchangeable apolipoproteins, including the kinetics of their adsorption and the physical properties of the interfacial layer.

Figure 1

(A) Surface pressure and (B) surface concentration (referred to as Γ_max) as a function of time of exchangeable apolipoproteins at the subphase concentration of 10⁻⁶ M. Open squares, ApoC-I; open circles, ApoA-I; solid circles, ApoA-II; solid triangles, ApoC-III; crosses, ApoC-II. (Insets) Comparison of native (open squares) and recombinant (solid squares) ApoC-I at the subphase concentration of 10⁻⁷ M.

Figure 2

BAM images of exchangeable apolipoproteins at the air-water interface after 5 h of adsorption at the subphase concentration of 10⁻⁶ M. (Left) Control (buffer); (right) protein. (A) ApoC-I; (B) ApoA-I; (C) ApoA-II; (D) Apo-CIII; (E) ApoC-II.

Figure 3

Apolipoprotein surface concentration (expressed in mol/m²). Solid bars correspond to the maximal surface concentration (Γ_max) reached at the end of the adsorption kinetics at the subphase concentration of 10⁻⁶ M. Hatched bars correspond to Γ₀, the minimal surface concentration necessary to initiate surface pressure (see Fig. 5 for the determination of Γ₀).

Figure 4

Surface pressure of ApoA-I reached at the end of the adsorption kinetics as a function of subphase concentration. (Inset) Surface pressure of other exchangeable apolipoproteins at the two subphase concentrations indicated by the arrows: 10⁻⁷ M (hatched bars) and 10⁻⁶ M (solid bars).

Figure 5

Estimation of Γ₀ and θ of exchangeable apolipoproteins calculated from the adsorption kinetics at the subphase concentration of 3 μg/ml, pH 7. (Open squares) ApoC-I; (open circles) ApoA-I; (solid circles) ApoA-II; (solid triangles) ApoC-III; (crosses) ApoC-II.

Figure 6

Surface concentration Γ (mg/m²), as a function of the square root of time, t^½, (h^½). (Open squares) ApoC-I; (open circles) ApoA-I; (solid circles) ApoA-II; (solid triangles) ApoC-III; (crosses) ApoC-II. In the first part of the kinetic adsorption at low subphase concentration the process is diffusion controlled. Hence, the surface concentration obeys the law: Γ = 2C_b(Dt/3.1416)^½;. The slope of the curve Γ versus t^½ (shown on the graph in case of ApoC-III) gives the value of the diffusion coefficient, D (m²/s⁻¹).

Figure 7

Shear elastic constant, μ, as a function of time of exchangeable apolipoproteins at the subphase concentration of 3 μg/ml. (Open squares) ApoC-I; (open circles) ApoA-I; (solid circles) ApoA-II; (solid triangles) ApoC-III; (crosses) ApoC-II. The end of the adsorption kinetics (∼10 h) is indicated by the arrow. (Inset) The mechanical response of the layer at the end of the kinetic interval was recorded as a function of the pulsation ω. The imaginary and real parts of the response adjust well to a harmonic oscillator, indicating that the monolayer fits an elastic layer model. For clarity, the imaginary part has been plotted versus −ω. The curves correspond to ApoA-I and are representative of the other exchangeable apolipoproteins.