Zakharenko O.K., Trusova V.M., Gorbenko G.P.

V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine

HEMOGLOBIN BINDING TO MODEL MEMBRANES

Elucidating the nature of hemoglobin (Hb) – lipid interactions seems to be of great importance in several main aspects. First, Hb-lipid systems provide a useful model for gaining insight into the mechanisms by which soluble proteins interact with biomembranes. Second, liposome-encapsulated Hb has found potential use as a red blood cell substitute. Third, Hb electrocatalytic and peroxidase activities are widely exploited in biosensing. In the present work we used several spectroscopic techniques to characterize lipid-associating and membrane-modifying properties of Hb. More specifically, our attention was focused on: i) Hb adsorption onto lipid bilayer, ii) protein conformational changes upon complexation with model membranes, and iii) Hb effect on physicochemical properties of lipid bilayer.

Fig. 1. (A) Elution profile of Hb-liposome mixture obtained by size-exclusion chromatography on Toyopearl HW-60F gel. (B) Hb adsorption isotherms onto phospholipid vesicles. Lipid concentration was 1 mM.

The first step of the study involves obtaining the adsorption isotherms by separation of Hb-liposome complexes and free Hb using gel filtration technique (Fig. 1). Lipid vesicles were prepared from zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL) with CL content 5, 10 and 20 mol%.

Hb complexes with lipids are stabilized by electrostatic and hydrophobic interactions, the latter favors protein penetration into the nonpolar region of membrane. The obtained binding data were quantitatively analyzed in terms of lattice model of large ligand adsorption to membranes allowing for the possibility of protein insertion into bilayer interior. Presented in Table 1 are thermodynamic parameters of Hb-lipid binding – association constant (K_a), free energy change (ΔG), number of lipid molecules per bound protein (n).

Table 1

Thermodynamic parameters of Hb-lipid binding

System	n	Ka, M^-1	ΔG, kJ/mol
PC	18.8±5.6	(1.8±0.5)×10^4	-24.3±7.3
PC:CL (5 mol% CL)	16.6±5.0	(2.4±0.7)×10^3	-19.3±5.8
PC:CL (10 mol% CL)	19.1±5.7	(4.5±1.4)×10^3	-20.8±6.2
PC:CL (20 mol% CL)	17.3±5.2	(6.7±2.0)×10^3	-21.8±6.5

The highest Hb affinity was found for neutral PC liposomes, suggesting the predominant role of hydrophobic binding component. CL-containing systems were featured by the lower association constants, although increasing with CL content.

Fig. 2. (A) Fluorescence spectra of rhodamine 101 in hemoglobin – liposome mixtures. (B) Hb effect on DSP-12 fluorescence intensity.

At the second step of the study we evaluated the possibility of employing rhodamine 101 (R101) as a specific fluorescent probe for detecting Hb conformational changes. The spectral parameters of R101 remained virtually unchanged in the presence of liposomes, suggesting that this probe either is insensitive to its transition from the aqueous to lipid phase or is incapable of associating with lipid bilayer. However, it appeared that R101 can easily associate with Hb as can be judged from R101 fluorescence intensity changes. Addition of isolated Hb-liposome complexes to R101 in buffer was accompanied by the changes in the shape of emission spectra suggesting the existence of several spectral bands and enhancement of shorter-wavelength (590 nm) component with increasing amount of membrane-bound Hb (Fig. 2A). These effects are most likely to arise from Hb structural changes at lipid-water interface.

At the last step of the study fluorescent probe DSP-12 was employed to obtain information about Hb effect on physicochemical properties of lipid bilayer. Hb-lipid binding was accompanied by the decrease of DSP-12 fluorescence intensity, with the magnitude of this effect being increased with CL content. Suppression of this effect in the presence of free radical scavenger thiourea allowed us to conclude that DSP-12 is sensitive to Hb-induced lipid peroxidation. Deconvolution of DSP-12 fluorescence spectra yielded two spectral components with emission maxima around 567 and 620 nm, which correspond to the probe populations differing in the location with respect to lipid-water interface. Relative contribution of these components proved to depend on Hb concentration and lipid composition of model membranes. More specifically, contribution of the shorter-wavelength component increases with CL content and Hb amount. These findings suggest that fluorescence of the longer-wavelength component adopting more shallow bilayer location is quenched by the polar products of free radical reactions, accumulating in the interfacial region.

Cumulatively, the present study revealed several new features of Hb-lipid interactions which may prove of interest both from fundamental and practical viewpoints.