Complexation of cytochrome c with calixarenes incorporated into the lipid vesicles and supported membranes

Abstract

We studied the interaction of cytochrome c (cyt c) with specific calixarenes (CX) incorporated into the large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) or supported lipid membranes (sBLM) and compared this with not specific adsorption of cyt c to the LUV containing DMPC and anionic phosphatidic acid (PA) or sBLM composed of a mixture of DMPC and dimyristoylphosphatidic acid (DMPA). We showed that with increasing concentration of CX the average size of LUV increased and zeta potential become more negative as it is suggested from dynamic light scattering experiments. For PA containing LUV the increase in vesicle diameter was less expressed, but zeta potential decreased similarly like that of LUV contained CX. Cyt c did not affect significantly the LUV size, but reduced the negative zeta potential both for CX and PA containing vesicles. Electrochemical impedance spectroscopy allowed us to determine binding of cyt c to sBLM contained CX or DMPA. In both cases we observed decrease of charge transfer resistance with increasing cyt c concentration. The analysis of binding process suggests that the main driving force for interaction of cyt c with sBLM is the negative surface charge.

Introduction

The lipid vesicles and supported bilayer lipid membranes (sBLM) are convenient models of biomembranes [1]. In particularly they are useful for study the mechanisms of interactions of proteins with the cell surface. In recent years the sBLM modified by receptors have also been extensively used in the fabrication of the sensing layer of various kinds of biosensors [2]. Among various types of tested receptors calixarenes are of a particular interest due to their sensitivity and selectivity to various compounds [3]. Calix[n]arenes are macrocyclic aromatic molecules, which originate from the coupling of phenols and aldehydes. The index [n] refers to the number of phenol aromatic cycles in the molecule. In a calixarene molecule, phenol subunits are bridged via methyl groups. This provides the characteristic vase-like shape of the calixarene molecule. Moreover, modification of the side groups of calixarenes allows one to prepare tailor-made receptors with high affinity for specific target molecules [4], [5]. Recently calixarene sensitive to cytochrome c (cyt c) has been demonstrated [6]. Cyt c is small hemoprotein (molecular weight 12.4 kDa) found in the intermembrane space of mitochondria. Under physiological conditions it is positively charged owing to lysine and arginine amino acid residues. Cyt c plays a dual role in living system. It participates in electron transport and is responsible for the activation of the apoptotic pathway through releasing from mitochondria into the cytosol [7]. Many studies have been focused on interaction of cyt c with lipid membranes (see [8] and references herein). In particularly it has been shown that cyt c induced transition of lamellar phase composed of phosphatidylcholine and cardiolipin into the hexagonal phase which is favorable for transport of cyt c through hydrophobic part of the membrane [8]. At the same time, the detection of endogenous concentration of cyt c is of high importance for diagnosis of possible pathological processes in the organism. The selective extraction of cyt c with calix[6]arene carboxylic acid derivative (^tOct[6]CH₂COOH) has been described by Oshima et al. [6]. They showed, that cyt c can be extracted from aqueous to the organic phase. This is due to 19 lysine residues at the cyt c surface terminated by –NH₃⁺ groups onto which the calix[6]arene has stronger affinity in comparison with previously reported calixarene receptors. The calix[6]arenes adsorb to cyt c surface by adopting the amino group into the cavity formed by 6 negatively charged carboxylic group connected to the phenols. However, in contrast with cyt c, lysozyme does not form stable complexes with calixarenes due to insufficient amount of lysines at the surface. So far the complexation of calix[6]arene with cyt c has been studied only in a bulk liquid phase [6]. However, due to hydrophobic butyl residues the calix[6]arene can be incorporated into the lipid layer of vesicles. This give opportunity to analyze complexation between the calix[6]arene and cyt c in a water phase. We can expect that incorporation of calixarene into the lipid vesicles should affect their size and surface potential. These values could also be affected by cyt c. These studies were not performed so far. Thus the calix[6]arene can serve as a recognition element in sensor for detection of cyt c. According to our recently published results, sBLM from soy bean phosphatidylcholine containing this calixarene functions as an efficient recognition element in the detection cyt c [9]. At the same time, cyt c adsorbs also to anionic vesicles or sBLM, which can give rise to non-specific interactions. However, a comparative study of the specific and non-specific interactions of cyt c with a calixarene modified vesicles and sBLM contained anionic species are still missing.

The aim of this work is to explore the interaction of cyt c with large unilamellar vesicles (LUV) and sBLM modified with a calixarene functionalized with carboxyl groups. At the same time, non-specific interactions of cyt c with LUVs or sBLMs including anionic compounds has been investigated. This approach allows clarifying the degree of specificity in the interaction of cyt c with LUVs and sBLMs modified with the mentioned type of calixarene. To this end the size and the zeta potential of LUVs has been determined by means of the dynamic light scattering method. The interaction of cyt c with calixarene or anionic lipid contained sBLMs has been characterized by electrochemical impedance spectroscopy (EIS).