Wus K. O., Kutsenko O. K., Yudintsev A. V., Trusova V. M., Gorbenko G. P.

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

BINDING OF NEW CYANINE DYE TO

FIBRILLAR AGGREGATES OF LYSOZYME

Fig. 1. Fluorescenñe intensity spectra of C1 free in solution and bound to fibrillar lysozyme

Fig. 2. The isotherm of C1 binding to fibrillar Lz. Protein concentration was 1.1 µM

         Due to a strong causative link between protein amyloidogenesis and development of so-called conformational diseases, such as type II diabetes, spongiform encephalopathies etc., correct detection of protein pathological aggregates represents one of the acute problems [1, 2]. In this regard, cyanine dyes, typically used for DNA detection, have very attractive properties: i) large Stokes shift; ii) high extinction coefficient; iii) very weak fluorescence in aqueous solution [3]. The aim of the present study was to evaluate the potential of novel cyanine dye C1 for identification of amyloid fibrils of small cationic protein lysozyme (Lz).

The first step of the study involved obtaining the adsorption isotherms by examining C1 spectral behavior in the absence and presence of fibrillar lysozyme, highly ordered aggregates with a core cross-β-sheet structure, in which β-strands run perpendicularly to the long axis of the fibril [2]. The asymmetric C1 dye was synthesized in the University of Sofia. Protein fibrils were prepared by dissolving Lz in deionized water with subsequent slow addition of ethanol to a final concentration of 80% and constant agitation at ambient temperature. Fluorescence spectra were obtained during titration of fibrillar Lz solution with C1, using Perkin Elmer LS-55 spectrofluorimeter.

As shown in Fig. 1, C1 binding to Lz aggregates resulted in two-fold increase in the fluorescence intensity. The resulted adsorption isotherm has bimodal character (Fig. 2) and was analyzed within the framework of two binding models – Langmuir and Hill [4]. The first one can be written as:

                                (1)

where ,  are the total concentrations of the dye and protein, respectively;  – the number of the dye binding sites per protein molecule, Ka is association constant, a – coefficient of proportionality.  Presented in Table 1 are thermodynamic parameters of C1-Lz binding (Langmuir), obtained by fitting the initial part of experimental curve by Eq.(1). Notably, the recovered stoichiometry was comparable with that reported for Thioflavin T [2]. The high Ka  is indicative of the dye binding to specific sites of the fibrils. The dye-protein complex is probably stabilized by H-bonds and hydrophobic interactions of C1 with β-sheets. Taken into account that the molecular dimensions of C1 (2.3×0.7 nm) are similar to those of Congo Red (2.6×0.9 nm), we assumed that these dyes have similar binding sites, i. e. the binding occurs in the grooves of amyloid fibrils [2].

Table 1. Quantitative parameters of the dye binding to fibrillar lysozyme

Binding model

Ka , M-1

 

Langmuir

6900

0.01

–

 

Hill

8.27

1.77

2.55

 

 

 

 

 

 

 

Next, sigmoidal part of the adsorption isotherm was analyzed using the Hill binding model. This model suggests absolute cooperativity of the dye-protein interactions:

                                                                                       (2)

where B – concentration of the bound dye,  is Hill parameter. Calculation of the binding parameters in terms of this model showed that Ka is 3 order of magnitude lower than in the case of specific binding (Table 1, Hill). The Hill parameter  is greater than 1,   reflecting the cooperativity of C1-fibril association [4]. This model assumes nonspecific saturation of sites. There was no saturation observed for adsorption, considered in terms of Hill binding model (Fig. 2).

         At the last step of the study C1 binding to amyloid fibrils was analyzed by titration of the dye solution with Lz. Fitting the experimental results by Hill model yielded the values of binding parameters close to those presented in Table 1. The grooves, abundant in the structure of amyloid fibrils, may serve as potential  specific binding sites for C1, while nonspecific ones may appear as a result of the dye self-association [2].

         In summary, the present study revealed a complex nature of interactions between cyanine dyes and lysozyme fibrils, which must be taken into account while using this class of fluorophores for amyloid detection and characterization. This work was supported by the grants from Science and Technology Center in Ukraine (Project number 4534) and Fundamental Research State Fund (Project number F28.4/007).

References

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2.   Minna G.  Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils – current status // J. Chem. Biol. – 2010. – V. 3. – P. 1 – 8.

3.   Deligiorgiev T. G., Zaneva D. A. A novel method for the preparation of monomethine cyanine dyes // Dyes and Pigments. – 1999. – V. 41 – P. 49 – 54.

4.   Cantor C., Shimmel P., Biological Chemistry. Part III. The behavior of biological macromolecules. – San Francisco: W. H. Freeman and company, – 1980. – 536 p.