Zhytnyakovskaya¹ O.A., Kutsenko¹ O.K., Trusova¹ V.M., Gorbenko¹ G.P., Kirilova² E.M., Kirilov² G.K., Kalnina² I.

¹V.N. Karazin Kharkiv National University, Ukraine

²Daugavpils University, Latvia

Sensitivity of newly synthesized lipophilic benzanthrone dye to the membrane environment

Nowadays many techniques use fluorescent dyes for noncovalent and covalent labeling of biological objects. Of particular importance in this regard are lipophilic fluorescent probes, designed to localize in a hydrophobic region within a biological specimen. Such lipophilic properties are inherent to benzanthrone dyes, which belong to the group of fluorescent molecular rotors, exhibiting intramolecular charge transfer state (ICT). Because of their bright fluorescence, excellent color characteristics and high thermo and photostability, benzanthrone probes are used as disperse dyes for textiles and polymers, daylight fluorescent pigments and laser dyes [1,2]. High sensitivity of these compounds to the environment provokes their use as fluorescent microenvironmental sensors, particularly, in the studies of membrane structure and protein-lipid interactions.

Fig. 2. Emission spectra of IBH in PC (A) and in PC:CL (10 %) liposomes (B). Excitation wavelength was 520 nm.

The present study was undertaken to evaluate the sensitivity of a newly synthesized benzanthrone dye, referred to here as IBH, to the changes in physicochemical properties of lipid bilayer. For this purpose, fluorescent spectroscopy technique was employed. First of all we evaluated the lipophilic properties of the dye IBH and its sensitivity to the membrane environment. Fluorescence spectra of this dye were recorded in buffer solution (5 mM Na-phosphate, pH 7.4) and liposomal suspension. Liposomes were prepared from phosphatidylcholine (PC) and its mixtures with cardiolipin (PC/CL) and cholesterol (PC/Chol) by extrusion technique. The typical fluorescence spectra of this dye are represented in Fig. 2

To characterize IBH-lipid interaction quantitatively, we determined the dye partition coefficient () for different lipid systems by analyzing the binding curves, presented in Fig.3.

	Table 1. Quantitative parameters of the dye-lipid binding
	System	Partition coefficient	Quantum yield
	buffer		0.003
	PC		0.13
	PC/CL (5 %)		0.06
	PC/CL (10 %)		0.03
Fig. 3. Fluorescence intensity increase as a function of lipid concentration	PC/Chol (30 %)			0.02

Analysis of the recovered partition coefficients (Table 1) shows that inclusion of anionic CL into PC bilayer gives rise to the increase of partition coefficient relative to the neat PC membrane. This effect can not be explained by electrostatic dye-lipid interactions because IBH is uncharged molecule. Hence, this may be related to structural features of PC/CL membrane. The conical shape of CL molecule induces a negative curvature strain, so that bilayer polar region becomes more accessible to water [3]. The increase of partition coefficient observed on CL inclusion in PC bilayer can result from the increased bilayer hydration which favors partitioning of the probe molecule into membrane. Moreover, inclusion of anionic CL into PC bilayer gives rise to the decrease of fluorescence quantum yield relative to the neat PC membrane. This effect can be explained by the higher level of CL oxidation (oxidative index~1), which favors enchanced water penetration into the membrane interior.

As seen in Table 1, Chol addition to PC model membranes resulted in the decrease of fluorescence quantum yield coupled with the increase of values. 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. On the other hand, such additional packing defects may give rise to the decrease of fluorescent quantum yield in PC/Chol liposomes.

To summarize, the present study revealed that the examined dye displays high lipid-associating ability. Partition coefficient of IBH was found to increase upon inclusion of anionic lipid cardiolipin and cholesterol into phosphatidylcholine bilayer. This can be effectively used for tracing the physicochemical properties of biological membranes.

REFERENCES

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3. Laan E., Killian J., Kruijff B. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile // Biochim. Biophys. Acta 2004.V. 1666. P. 275-288.