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RESEARCH Fluorescent Probes Fluorescent dyes for study of beta-pleated protein formations

Fluorescent dyes for study of beta-pleated protein formations

Uncontrolled proteins aggregation is considered to be the molecular basis for a range of neurodegenerative diseases, among which Alzheimer's, Parkinson's and Creutzfeldt–Jakob diseases.

We proposed cyanine dyes as a new prospective class for development of fluorescent probes for detection and studies of protein aggregation [1,2].

Thus mono- (T-284) and trimethine (SH-516, 7519) cyanines (Fig. 1) allow quantitative detection of as little as ~1,5 µg/ml of fibrillar alpha-synuclein (ASN), a comparable detection limit with that of commercially available dyes [3].

Figure 1. Amyloid sensitive cyanine dyes
Figure 1. Amyloid sensitive cyanine dyes

For the detection of ASN oligomeric aggregates, which contain both beta-sheet and alpha-helical structural fragments, the dyes with a longer polymethine chain and bulky aromatic substituents, i.e. tri- (21252) and pentamethine (Sl-631) cyanines are proposed. For these dyes (Fig.2) the emission increasing in dozen times the presence of oligimeric spices was observed [4].

Figure 2. Cyanine dyes sensitive to oligomeric aggregates.
Figure 2. Cyanine dyes sensitive to oligomeric aggregates.

The data of fluorescent, fluorescence polarization, and fluorescence lifetime spectroscopy, as well as atomic force microscopy carried out together with Prof. V. Subramaniam (University of Twente, the Netherlands) allowed us to get insight into the mechanism of cyanine dye/amyloid fibril complex formation [5].

Figure 3. Zirconium phthalocyanine containing lysine fragments as out-planed substituents.
Figure 3. Zirconium phthalocyanine containing lysine fragments as out-planed substituents

The aggregation pathway of proteins into the amyloid fibrils is considered as the obvious target for therapeutic intervention in aggregation related diseases. The fluorescent assay for search of inhibitors of amyloid fibril formation was developed on the base of trimethinecyanine dye 7519 (Fig. 1).

It was successfully applied in study of effect of flavonoids on insulin aggregation in vitro. Besides, using this assay the high inhibitory activity of metal containing spatial complexes- phthalocyanines with out-planed ligands (Fig.3) and iron clathrochelates in fibril formation was demonstrated [6].


  • K.D. Volkova, V.B. Kovalska, A.O. Balanda, R.J. Vermeij, V. Subramaniam, Yu.L. Slominskii and S.M. Yarmoluk. Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures // Journal of Biochemical and Biophysical Methods. – 2007. – Vol.70, Iss. 5. – P.727-733.
  • K.D. Volkova, V.B. Kovalska, A.O. Balanda, M.Yu Losytskyy, A.G. Golub, R.J. Vermeij, V. Subramaniam, O.I. Tolmachev and S.M. Yarmoluk. Specific fluorescent detection of fibrillar α-synuclein using mono- and trimethine cyanine dyes/ Bioorganic and Medicinal Chemistry. – 2008. – V. 16, Iss. 3. – P.1452-1459.
  • K.D. Volkova, V.B. Kovalska, G.M. Segers-Nolten, G. Veldhuis, V. Subramaniam, S.M. Yarmoluk. Explorations of the application of cyanine dyes for quantitative α-synuclein detection // Biotechnic & Histochemistry. – 2009. – 84. – P.55‑61.
  • V. B. Kovalska, M. Yu Losytskyy, O. I. Tolmachev, Yu L. Slominskii, G. M. J. Segers-Nolten, V. Subramaniam and S. M. Yarmoluk Tri- and Pentamethine Cyanine Dyes for Fluorescent Detection of α-Synuclein Oligomeric Aggregates // Journal of Fluorescence. – 2012. – DOI: 10.1007/s10895-012-1081-x
  • K.D. Volkova, V.B. Kovalska, M.Yu. Losytskyy, G. Veldhuis, G. M. J. Segers-Nolten, O.I. Tolmachev, V. Subramaniam, S.M. Yarmoluk. Studies of Interaction Between Cyanine Dye T-284 and Fibrillar Alpha-Synuclein // Journal of Fluorescence. – 2010. – Volume 20, N6. – P.1267-1274.
  • V. Kovalska, M. Losytskyy, V.Chernii, K.Volkova, I.Tretyakova, V.Cherepanov, S.Yarmoluk, S.Volkov Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands// Bioorganic and Medical Chemistry. – 2012. – Vol. 20. – P. 330–334.

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