Ogurtsova¹ V.V., Zhytniakivska¹ O.A., Trusova¹ V.M., Gorbenko¹ G.P,

Kirilova² E.M., Kirilov² G.K., Kalnina² I.

¹V.N. Karazin Kharkiv National University, Ukraine

²Daugavpils University, Latvia

Interactions between a new fluorescent benzanthrone dye and model membranes

Due to their versatility luminescent techniques are widely used in biophysics studies, particularly for the covalent and noncovalent labeling of biological objects, including natural and model membranes. High sensitivity of fluorescent probes to the environment provokes strong prerequisites for their use as markers in probing the membrane structure and protein-lipid interactions. Among the organic luminophores, which localize in the hydrophobic region of liposomes are benzanthrone dyes composed of 3-methoxybenzanthrone. Because of their bright fluorescence, and color characteristics these probes are widely used as luminescent pigments and daylight components in lasers [1, 2].

Fig 1. Typical emission spectra of IAH in PC (A) liposomes and PC/Chol (30%) liposomes (B). Excitation wavelength was 520 nm.

The purpose of this work was to investigate the sensitivity of a new benzanthrone dye, referred here as IAH, to the changes in membrane environment. For this purpose the method of fluorescence spectroscopy was used. Firstly the partition coefficients of the dye in the lipid phase were measured by titration of the probe IAH with liposomes, which composed of phosphatidylcholine (PC) and its mixtures with cholesterol (PC/Chol) and cardiolipin (PC/CL). Liposomes were prepared by extrusion technique [3]. The typical fluorescence spectra of this dye are represented in Fig.1.

To characterize IAH-lipid interaction more detail, we determined the dye partition coefficients (K_p) for different lipid systems by analyzing the binding isotherms, presented in Fig.2.

	Table 1. Quantitative parameters of the dye-lipid binding
	System	Partition coefficient	Quantum yield
	PC	3474±578	0.06
	PC / CL 5%	6556±380	0.05
	PC / CL 10%	1464±236	0.04
Fig.2. Fluorescence intensity increase as a function of lipid concentration	PC / Chol 30%	5584±868	0.07

As seen in Table 1, inclusion of sterol Chol into PC bilayer give rise to increase K_p and fluorescence quantum yield relative to the neat PC membrane. Such effects can be interpreted in terms of the appearance of additional packing defects in the interfacial bilayer region on Chol addition. It is assumed that the changes in lipid packing density on Chol inclusion allow a greater number of water molecules to penetrate in the headgroup bilayer region, which, in turn, brings about the increase of partition coefficient compared to the neat PC membrane.

In CL-containing systems partitioning coefficient was found to exhibit unambiguous behavior (it has a tendency to increase in PC/CL (5%) and decrease in PC/CL (10%)), when the fluorescence quantum yield of dye IAH decrease relatively to the neat PC membrane. Such quantum yield decrease can be explained by the higher level of CL oxidation (oxidative index~1), which favors enchanced water penetration into the membrane interior. Unambiguouse behavior of K_p in CL-containing systems can be interpreted in terms of specific conical structure of CL molecule.

At the next step of the study the fluorescence anisotropy of IAH in different lipid systems were measured by adding to the liposome-containing systems a native protein lysozyme. Present study shows that inclusion of lisozyme to PC/CL (10%) membrane give rise to increase of the fluorescence anisotropy of the dye IAH. It can be explained in terms of electrostatic interactions between the anionic lipid cardiolipin and opposite charged lisozyme.

Fig.3 Fluorescent anisotropy after addition of native protein lisozyme.

In conclusion, the present study demonstrated that the examined dye IAH displays high lipid-associating ability. It was found that partition coefficient of IAH increases upon inclusion of cholesterol into phosphatidylcholine bilayer. The obtained results suggest that benzanthrone dyes can be effectively used as markers of physicochemical properties of the biological objects.

References

1. Dobretsov G.E. Fluorescent probes in studying cell membranes and lipoproteins//M.:Nauka.1989.

2. Vladimirov Y.A., Potapenko A.Y. Physico-chemical bases of photobiological processes //M.: Drofa, 2006.

3. Mui B., L. Chow L., Hope M.J. Extrusion technique to generate liposomes of defined size //Meth. Enzymol. 2003. V. 37, P. 3-14.