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

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

Cholesterol effect on hemoglobin-lipid interactions

Cholesterol (Chol) effect on hemoglobin (Hb) binding to lipid bilayer is a problem attracting considerable attention. In particular, specific sterol location in phospholipid bilayers stabilizes Hb structure, prevents protein oxidation, dissociation and loss of its physiological activity [1]. Furthermore, it was recently found that cholesterol, associated with phospholipids, is capable of forming complexes with Hb in human erythrocytes. Although these complexes exert influence on the cell antioxidant system, they do not directly interact with reactive oxygen species. The role of Hb-Chol complexes is thought to involve the shifts of correlations in the erythrocyte defense system [2,3].

Fig. 1. Elution profile of Hb-liposome mixture	Fig. 2. Hb-lipid adsorption isotherms. Lipid concentration 1 mM.

The present work was focused on examination of Hb binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol). Hb adsorption onto lipid bilayer was studied using the size exclusion chromatography on the gel Toyorearl HW-60F. Fig. 1 represents typical elution profile of Hb-lipid mixtures, in which the first peak corresponds to the protein-liposome complexes and the second peak – to the protein free in solution. A set of such profiles obtained for different Hb concentrations yielded adsorption isotherms presented in Fig. 2. Experimental binding curves were then quantitatively analyzed in terms of the lattice model of large ligand adsorption to membranes allowing for the possibility of protein insertion into bilayer interior (Eqs. 1-2).

, Eq. 1

, , , Eq. 2

where K_a – association constant, F – concentration of Hb free in solution, B – concentration of lipid-bound Hb, L – lipid concentration, n₁, n₂ – the number of lipid molecules constituting the binding sites for surface-bound and bilayer-inserted Hb, respectively, n=n₁+n₂ – total number of lipids per bound protein, z = 6 – coordination number for hexagonal lattice, ΔG – free energy change, R – universal gas constant, T – temperature.

This thermodynamic model takes into account specific features of protein-lipid complexation, such as large size of the protein, ability of Hb to form contacts with several lipids, and steric area-excluding interactions between absorbed protein molecules. Analysis of the data presented in Table 1 suggests that Chol may affect both Hb-lipid binding energy and stoichiometry.

Table 1

Thermodynamic parameters of Hb-lipid binding

System	n	Ka, M^-1	ΔG, kJ/mol
PC	17±2,5	(1,5±0,2)×10^4	-23,8±3,6
PC:Chol (5 mol% Chol)	33±5,0	(1,9±0,3)×10^4	-24,4±3,7
PC:Chol (15 mol% Chol)	20±3,0	(3,8±0,6)×10^3	-20,4±3,1
PC:Chol (30 mol% Chol)	33±5,0	(2,5±0,4)×10^4	-25,1±3,7

Several lines of evidence indicate that sterol effect on physicochemical properties of a lipid bilayer may involve: i) increased phospholipid headgroup separation, ii) increased freedom of motion of phosphorylcholine moiety, iii) ordering of lipid hydrocarbon chains (condensing effect). These bilayer perturbations may be coupled with Chol-mediated Hb conformational changes, protein oxidation and dissociation of heme-globin complex. Such a behavior may manifest itself in the increased constant of Hb binding to model membranes containing 30 mol% of Chol as compared to neat PC bilayer.

Non-monotonous dependence of K_a changes on Chol content may be rationalized in terms of the lattice model of lipid lateral distribution. Briefly, this model implies the variations in membrane free volume in two-component lipid bilayers stemming from the difference in cross sectional areas between the guest (Chol) and matrix (PC) lipids. The extent of membrane free volume fluctuations varies periodically with mole fraction of the guest lipid. It may be assumed that these free volume fluctuations underlie the nonlinear dependence of binding parameters on Chol concentration.

This work was supported by the Grant number 4534 from the Science and Technology Center in Ukraine.

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