Methemoglobin binding to model phospholipid membranes

The interaction of methemoglobin with model phospholipid membranes composed of phosphatidylcholine and its mixtures with phosphatidylserine or diphosphatidylglycerol has been studied. The binding isotherms have been analyzed in terms of two-dimensional lattice models of surface adsorption and incorporation of the protein into the lipid bilayer. The binding parameters including an association constant, binding stoichiometry, enthalpy and entropy contributions to free energy change have been estimated.

Introduction.Model protein-lipid systems are pre sently widely used to gain insight into the nature of interactions between two major membrane consti tuents-proteins and lipids [1,2].Protein compo nent of these systems is often represented by nonmembraneous water soluble proteins, being capable of forming complexes with lipids [3,4].One of the proteins employed in such model studies is hemo globin [5].
Numerous data available in the literature are indicative of the possibility of hemoglobin binding to lipid bilayer by means of electrostatic and hydro phobic interactions [5-7 J.To date, hemoglobin stru cture is well characterized, thus providing a basis for elucidation of general principles and driving forces of protein-lipid interactions.One important aspect of the problem envisaged concerns the factors governing the thermodynamics of the protein association with a lipid bilayer.
The main goal of the present work was to examine thermodynamic characteristics of methemo globin (metHb) complexes with phospholipids.Analy sis of the binding isotherms in terms of two-dimen sional lattice models of large ligand adsorption to membranes allowed to estimate association constant, stoichiometry of binding, enthalpy and entropy cont ributions to the free energy change.As a lipid component of the model systems studied liposomes composed of phosphatidylcholine (PC) and its mixtu-res vrith phosphatidylserine (PS) and diphospha tidylglycerol (DPG) were employed.
Materials and Methods.Egg yolk PC, beef heart DPG and beef brain PS were purchased from Bakpreparat (Kharkiv, Ukraine).Oxyhemoglobin was isolated from human blood according to [8] and converted to metHb by adding of potassium ferricyanide with subsequent gel-filtration on molselect G-25.Liposomes from PC and its mixtures with PS (3:1, mohmol) and DPG (6:1, mohmol) were obtained as follows.Ethanol lipid's solution was evaporated under vacuo and lipid film was then suspended in 10 mM Tris-HCl buffer, pH 7.4, to a final lipid concen tration of 10 mg/ml.lipid suspension was shaked for 10 min, sonicated for 3 min, 4 °С and centrifuged at 30000 g for 30 min in order to remove multilamellar vesicles.Liposomes with average diameter of ca. 100 nm, remaining in supernatant, were used in expe riments.Phospholipid concentration was determined according to [9].Protein concentration was found using extinction coefficient E 401 = 5.66-10 5 M _I cm~ [101.
Results and Discussion.In analyzing the protein (P) association with lipids (L) one of the following approaches is commonly used.Within the framework of traditional approach lipid bilayer is treated as consisting of discrete protein's binding sites, each containing n lipid molecules.In this case binding curves; are described by Langmuir isotherm, being applied to a simple chemical equilibrium: P + nL ** о P-«L.However, as indicated in a number of studies [11][12][13][14], this approach appears to be inade quate in examining protein-lipid interactions for the following reasons.First of all, the protein must be considered as large ligand, interacting with an array of binding contacts on a membrane surface and covering simultaneously n lipid molecules.Additio nally, adsorption of large ligand on the surface can lead to the steric constraints depending on the spatial arrangement of the lipids in the protein-lipid contact region.
Such peculiarities of the protein interaction with lipid bilayer have been taken into account in a series of models, particularly, in the models proposed by Stankowski [12,13].It seems also noteworthy that there exists an approach, principally differing from those mentioned above.It based on the consideration of the protein association with an assembly of lipids, rather than with individual molecules comprising lipid bilayer, so that protein-lipid interactions are inter preted as partitioning of the protein between aqueous and membrane phases, characterizing only by parti tion coefficient [14].
In the present paper two-dimensional lattice models of Stankowski [12,13] have been used to analyze quantitatively metHb binding to liposomes of various composition.Lipid bilayer was modeled as hexagonal lattice with structural subunits represented by lipid molecules.According to the formalism emp loyed, two limiting cases, corresponding to the linear and discoid ligand shape, were considered.Note that the concept of «ligand shape» means geometrical arrangement of binding contacts in the protein-lipid complex.Adsorption of linear ligand on the mem brane surface was described by [13]: where B, F are the concentrations of bound and free protein, respectively, L is the total lipid concentration, n is the number of lipid molecules per molecule of bound protein, K a is association constant, z is the lattice coordination number (z = 6).Assuming that ligand has a shape of disc the following relationship was used: (3) where a is the parameter of excluded area (a = 3), со = 2v r 3n7#.
Equations ( 1 (Fig. 1, 2).In this case it was assumed that the main type of protein-lipid interactions is the adsorption of the protein molecule on membrane surface due to formation of electrostatic contacts.Meanwhile, taking into account the findings provided by a number of studies [6,7,15], preferential mode of metHb interaction with neutral PC vesicles was supposed to be the protein penetration into bilayer interior.The refore the binding curves observed for PC liposomes (Fig. 3) were treated in terms of the model of protein incorporation in the membrane [13]: Parameter n in eqn.( 4) corresponds to the number of lipid molecules occupying surface area being equivalent to cross-section of the protein part penetrating in the bilayer.
The binding of metHb to liposomes was exa mined by monitoring the decrease of protein absor- bance in Soret band (at wavelength 407 nm).This absorbance change is supposed to be a consequence of metHb structural alterations caused by its interaction with lipid's hydroperoxides [16] or negatively char ged phospholipids [17,18].According to approach, developed in the previous studies [20,21 ], it was assumed that absorbance decrease (АЛ 407 ) is pro portional to the concentration of the bound protein (B): where a is coefficient of proportionality.The mea sured value of (Ai4407) (AA r ) was corrected for the light scattering of the protein-lipid mixture using the following relationship [20]: where A 0 is the wavelength from the region of neglijjibly small protein absorbance (A 0 = 700 nm), A = 407 nm.Parameter m, determined by the sample turbidity, was estimated from the plots lg Л vs. lg A obtained at wavelengths 600-700 nm, according to equation: where P Q C and P 0 e are calculated and determined experimentally concentrations of the protein, m is the number of experimental points.
Presented in Table 1 are the parameters n and K a characterizing metHb association with liposomes of various composition.Since the ligand shape is un known and proves to be irregular, the lower and upper limits of n were assessed assuming linear or discoid ligand shape, respectively.Because the crosssection of metHb molecule being ca.2600 A, corres ponds; to the area of ca.37 lipid molecules, it seems likely that real shape of contact region in the protein-

Table 1 Parameters of methemoglobin binding to liposomes derived from the fitting of experimental data to eqns. (l)-(6)
The error of parameter estimation does not exceed 17 % for K a 20 % for n and 12 % for a lipid complexes is closer to linear.It should be emphasized that parameter n accounts for a total amount of lipid molecules that are excluded from the further ligand binding upon the adsorption of one protein molecule.The actual number of protein-lipid contacts in some cases can be less than n.Such a situation may take place, for instance, when the protein binds preferentially to the charged lipid headgroups, that form clusters upon the protein association with lipid bilayer [13].Taking into ac count this possibility, relatively high n values, derived for liposomes PC:PS (Table 1) can be interpreted in terms of preferential metHb binding to negatively charged PS molecules.In this case the number of lipids in contact with the protein (n*) can be eva luated as nf, where / is the fraction of charged lipid (f = 0.25).The values of n*, obtained in such a way, would be closer to aforementioned estimates, based on the protein cross-section and the area of lipid's headgroup.
The value of association constant (Table 1) observed at different temperatures were further used to estimate free energy change (AG) and its enthalpy (AH) and entropy (AS) contributions: The fitting of In K a plots vs. 1/T to eqn. ( 12) allowed to evaluate AH and AS.As can be seen from Table 2, the formation of metHb complexes with liposomes is characterized by positive enthalpy and entropy changes.The values of AG are consistent with those reported elsewhere for model peptides [19] and proteins [22].According to the modern theories of protein-lipid interactions there exist at least five factors controlling the thermodynamics of the protein binding to lipid bilayer, namely i) formation of electrostatic contacts, ii) hydrophobic effect and alte rations in iii) the network of hydrogen bonds, iv) protein entropy and v) lipid ordering [23 ].As follows from the theoretical predictions, changes in hydrogen Table 2 Thermodynamic parameters of methemoglobin interaction with liposomes bonding and lipid ordering do not contribute no ticeably to the total AG value.However, another factors afore-mentioned may be of significance.The results presented here suggest that the main driving force of metHb interaction with lipids is energetically favourable entropy increase.This, in turn, can be caused by i) formation of ionic contacts in water, ii) transfer of nonpolar amino acid sidechains in the membrane interior and iii) unfolding of the protein molecule [23].In this context it seems important to note that negatively charged lipids, including PS and DPG, can exert destabilizing influence on metHb structure [17,18].
Taken together, the results of the present study indicate that two-dimensional lattice models allow to obtain reasonable estimates of the binding para meters, characterizing either surface adsorption or protein incorporation in the lipid bilayer.From thermodynamical viewpoint, entropy factor was found to play determining role in metHb association with the model phospholipid membranes.
Acknowledgements.I thank the referee for the valuable remarks.