Studies of Interaction Between Cyanine Dye T-284 and Fibrillar Alpha-Synuclein

A key feature of Parkinson’s disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the “binding channel” running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different “populations” due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.     

Tri- and Pentamethine Cyanine Dyes for Fluorescent Detection of α-Synuclein Oligomeric Aggregates

The pathogenesis of Parkinson’s disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10^?6 M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2–2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more “crescent-shape” molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.     

Fluorescence Study of the Membrane Effects of Aggregated Lysozyme

The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp₆₂ and Trp₁₀₈ increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.     

Fluorescence Characteristics of Variously Charged Asymmetric Monomethine Cyanine Dyes in the Presence of Nucleic Acids

Three asymmetric monomethine cyanine dyes bearing one, two, and three positive charges have been synthesized, and their absorption and fluorescence characteristics in the presence of nucleic acids were studied. The maxima of their longest wavelength absorption band lie between 500 and 520 nm. The dyes do not show fluorescence of their own in TE buffer (pH = 7.5), but become strongly fluorescent (Q_F = 0.2–0.6) on binding to double-stranded DNA. The fluorescence maxima of the investigated dye-dsDNA complexes are in the region of 530–550 nm. The influence of the dye/DNA ratio on both the position and intensity of the fluorescence maxima of the complexes is investigated.     

Hydroxy and Methoxy Substituted Thiacarbocyanines for Fluorescent Detection of Amyloid Formations

In present paper series of trimethine cyanines modified in 5,5′- or 6,6′- position with hydroxy- or methoxy- substituents is studied for their ability to interact selectively with fibrillar formations. Processes of dye aggregation that accompany this interaction were also investigated. Meso-methyl trimethynecyanines with 5,5′- methoxy (7519) and hydroxy (7515) substituents strongly (up to 40 times) increase fluorescence intensity in the presence of fibrillar insulin, and also give noticeable fluorescent response on the presence of various aggregated proteins (lysozyme, β-lactoglobulin, α-synuclein A53T). 7519 and 7515 dyes can be used for fluorometric detection of fibrillar insulin at concentrations of approximately 1.5–120 microg/ml. For meso-ethyl substituted dye 7514 the ability to form H- and J-aggregates upon interaction with insulin fibrils was suggested. The model of the H- and J-aggregate packing in the protein fibrillar structure has been proposed.     

Effect of hydrophobicity of cationic carbocyanine dyes DiOC<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> on their binding to anionic surfactant micelles

The interaction of a series of cationic dialkyloxacarbocyanine perchlorate (DiOC _n ) dyes of different degrees of hydrophobicity with micelles of an anionic surfactant, sodium dodecylsulfate (SDS), has been studied spectrophotometrically in aqueous solutions. The Benesi–Hildebrand equation was used to calculate binding constants (K _b ) of the dyes to surfactant micelles, the fraction of dye bound to the micelles (f _mic ), and the standard free-energy change (ΔG ⁰) for the transfer of dye from the aqueous to micellar phase. It has been shown that the interaction of oppositely charged dye molecules and surfactant micelles is controlled by both electrostatic and hydrophobic interactions. A small increase in dye hydrophobicity due to lengthening of the hydrocarbon radical has been shown to cause an abrupt nonlinear increase of the fmic value. This points to a key role of hydrophobic interactions in the binding of dye molecules with the micelles.     

Hydrophobicity effect on interactions between organic molecules in nanocages of surfactant micelle

Interactions between the cationic polymethine dyes 3,3′-diethyloxacarbocyanaine iodide (DiOC₂), 3,3′-dioctadecyloxacarbocyanine iodide (DiOC₁₈), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) in the nano-scale volume of anionic sodium dodecylsulfate (SDS) micelles have been studied using optical spectroscopy techniques. The solubilization of pairs of dye molecules (DiOC₁₈/DiI or DiOC₂/DiI) within SDS micelles was monitored by Forster resonance energy transfer (FRET) between the dyes in each pair. The influence of the hydrophobicity of the dyes DiOC₂ and DiOC₁₈ on the efficiency of their binding to SDS micelles and, consequently, on the efficiency of FRET between DiOC₁₈ and DiI or DiOC₂ and DiI fluorophors has been analyzed. It has been shown that hydrophobic interactions in addition to electrostatic interactions are of key importance for the binding between the positively charged dyes and negatively charged surfactant micelles. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 646–652, September–October, 2008.     

Synthesis and Spectra of a Kind of Novel Longer-Wavelength Benzoxazole Indole Styryl Cyanine Dye with a Carbazole-Bridged Chain

Based on cyanine dye probe oxazole yellow (YO) and Cy₃, a series of novel styryl cyanine dyes were designed and synthesized. Carbazole was inserted into the structures of YO and Cy₃ to act as a bridge to link the benzoxazole and indole group. This modification resulted in a novel kind of benzoxazole indole styryl cyanine dye with a carbazole-bridged chain. The dyes were characterized by ¹HNMR and MS. The spectra of the novel dyes were also performed and the results showed that the maximum emission wavelength of the carbazole styryl cyanine dye was shifted red, the Stokes shift increased and the fluorescence intensity enhanced compared with those of YO and Cy₃. These results indicated that the novel dye could be used as an excellent fluorescent probe in biological labeling.     

Luminescence properties of pyron red—A new molecular probe for protein research

The dependence of the luminescence of the new anionic dye Pyron Red (PR) on the polarity of the medium is investigated. Upon passage from an aqueous phase to a nonpolar phase, PR shows a shortwave shift of the fluorescence emission maximum from 675 to 650 nm and an increase in the fluorescence quantum yield from 0.03 to 0.54–0.70. When complexed with human serum albumin, PR shows fluorescence excitation and emission maxima at 525 and 625 nm and a fluorescence quantum yield of 0.8. In a comparison of the luminescence properties of PR with those of the well-known probes ANS and K35 in water and a complex with albumin, PR is shown to have the maximum absolute sensitivity but a lower fluorescence enhancement upon binding with a protein compared to ANS. A convenient criterion of the probe sensitivity toward binding with a protein that is defined as the ratio of the fluorescence intensities of the protein-bound and the free probe A_F=F_b/F_f is proposed. The value of A_F(35) for the PR probe ranks between those for the K35 probe with a low A_F(18) and ANS with a high A_F(105). Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 66, No. 3, pp. 369–374, May–June, 1999.     

Photophysical studies of PET based acridinedione dyes with globular protein: Bovine serum albumin

Interaction of acridinedione dyes with model transport proteins, bovine serum albumin (BSA) in aqueous solution were investigated by fluorescence spectral studies. A fluorescence enhancement was observed on the addition of BSA to photoinduced electron transfer (PET) based acridinedione dyes, which posses C₆H₄(p-OCH₃) in the 9th position of the basic acridinedione ring. On the contrary, the addition of BSA to non-PET based acridinedione dyes with methyl or phenyl substitution in the 9th position does not result in any fluorescence enhancement. The enhancement in the fluorescence intensity is attributed to the suppression of PET process through space between -OCH₃ group and the acridinedione moiety is elucidated by steady state fluorescence measurements. The fluorescence anisotropy value (r) of 0.40 reveals that the motion of the dye molecule is highly constrained and is largely confined to the rigid microenvironment of the protein molecule. The binding constant (K) was found to be in the order of 6.0×10³ [M]⁻¹, which implies the existence of hydrophobic interaction between the PET based dye and BSA. Time resolved fluorescence lifetime measurements reveal that the PET based acridinedione dye preferably binds in the hydrophobic interior of BSA.     

Material properties controlling photocurrent on TiO2 aggregates with plane orientation for dye-sensitized solar cells

Effective design of a dye-sensitized solar cell (DSSC) requires a clear understanding of the reaction mechanisms of the constituent cell materials. The relationship between structural and photo-electrochemical properties of the photo-anodic materials is of the first priority for such investigations. Highly oriented aggregates of anatase phase TiO₂ nanoparticles were deposited on Indium Tin oxide (ITO) glass substrates; over which N719 dyes were adsorbed through electrophoretic deposition under a strong magnetic field. The properties were evaluated by electrochemical measurements, UV–VIS spectroscopy, and electrical resistance measurements. The results showed that the absorbed photon number in the TiO₂ aggregates with adsorbed dye and their resistivity showed different dependences on the orientation of the crystal plane in the TiO₂ particle. The dependence of the photocurrent on the plane orientation of aggregates of dye-sensitized TiO₂ nanoparticles has been determined from a combination of the electrical conductivity of TiO₂ aggregate and the amount of dye adsorbed on the surface of TiO₂.     

The Mechanism of Benzothiazole Styrylcyanine Dyes Binding with dsDNA: Studies by Spectral-Luminescent Methods

In the presented work studies of the interaction mode of monomer and two homodimer benzothiazole styryl dyes containing spermine-like linkage/tail group with the double stranded (ds) DNA are reported. For these dyes, equilibrium constant of dye binding to DNA (K _b), as well as the number of dsDNA base pairs occupied by one bound dye molecule (n) were determined. The data obtained show that the presence of spermine-like group containing quaternary nitrogen (Bos-5) results in increase of K _b value as compared to this of unsubstituted analogue (Sbt). Besides, for the dimer dyes containing benzothiazole styryl chromophores, the K _b value is either five times higher (DBos-13) or almost the same (DBsu-10) as compared to this of corresponding monomer Sbt, depending on the position in the benzothiazole ring where the linker is attached. Moreover, the n values for both dimers are significantly different as well, pointing to the bis-intercalative binding mechanism for DBos-13 and for the groove-binding one for DBsu-10. The conclusion about the dimer dyes-dsDNA binding mechanisms is also supported by the study of the fluorescent response of these dyes on the presence of AT- and GC-containing polynucleotides.     

Unfolding and Fibrillogenesis of Insulin: Temperature,Pressure and Chemistry

Protein folding or unfolding can lead to the population of intermediates or partially unfolded conformations that have a high aggregation tendency. Some of these states associate in vivo to form fibrillar structures. These fibrils are the hallmark of molecular diseases such as Alzheimer's disease. It has been suggested that in vitro fibril formation is a generic property of all proteins. Insulin has been chosen as a model protein to study the process of fibrillation with Fourier-transform infrared spectroscopy. It is found that the formation of fibrils is preceded by amorphous aggregation. We also investigated the effect of hydrostatic pressure on insulin fibrils. The observed spectral changes are interpreted in terms of fibril dissociation into protofilaments. Preliminary results indicate that pressure is an interesting tool to characterize the interactions that maintain the fibril structure.     

Protein adsorption drastically reduces surface‐enhanced Raman signal of dye molecules

There is an increasing interest in developing surface enhancement Raman spectroscopy methods for intracellular biomolecule and for in vitro protein detection that involve dye or protein–dye conjugates. In this work, we have demonstrated that protein adsorption on silver nanoparticle (AgNP) can significantly attenuate the surface‐enhanced Raman spectroscopy (SERS) signal of dye molecules in both protein/dye mixtures and protein/dye conjugates. SERS spectra of 12 protein/dye mixtures were acquired using 4 proteins [bovine serum albumin (BSA), lysozyme, trypsin, and concanavalin A] and three dyes [Rhodamine 6G, adenine, and fluorescein isothiocyanate (FITC)]. Besides the protein/dye mixtures, spectra were also obtained for the free dyes and four FITC‐conjugated proteins. While no SERS signal was observed in protein/FITC mixtures or conjugates, a significantly reduced SERS intensity (up to 3 orders of magnitude) was observed for both R6G and adenine in their respective protein mixtures. Quantitative estimation of the number of dye molecules absorbed onto AgNP implied that the degree of R6G SERS signal reduction in the R6G/BSA sample is 2 to 3 orders of magnitude higher than what could be accounted for by the difference in the amount of the absorbed dyes. This finding has significant implications for both intracellular SERS analyses and in vitro protein detection using SERS tagging strategies that rely on Raman dyes as reporter molecules. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and properties of magnetic and luminescent Fe3O4/SiO2/Dye/SiO2 nanoparticles

A simple and reproducible method was developed to synthesize a novel class of Fe₃O₄/SiO₂/dye/SiO₂ composite nanoparticles. As promising candidates for use in bioassays, the obtained nanoparticles have an average diameter of 30 nm, and the thickness of the outer shell of silica could be tuned by changing the concentration of the silicon precursor tetraethyl orthosilicate during the synthesis. These multifunctional nanoparticles were found to be highly luminescent, photostable and superparamagnetic. The luminescence intensity of the nanoparticles was increased as the dye concentration was increased in the preparation process. The color of the luminescence was successfully tuned by incorporating different dyes into the nanoparticles. The measurements of the emission spectra indicated that relative to the dye molecules dissolved in ethanol, the emission of the dye-doped nanoparticles exhibited either a red shift or a blue shift, to which a tentative explanation was given.     

Computational study of new azo dyes with different anchoring groups for dye-sensitised solar cells

Some of new azo dyes with different anchoring groups, such as biscarbodithiolic acid, hydroxamic acid, phosphonic acid, carboxcylic acid and sulfonic acid have been investigated theoretically to evaluate the effects of various anchoring groups on the optical and electronic properties of the dyes in dye-sensitised solar cells. Optical and electronic properties, UV–Vis absorption spectra, light-harvesting efficiency, lifetime of the excited state, chemical hardness and lowest unoccupied molecular orbital (LUMO) orbital weight of the dyes on the anchoring groups, have been studied to shed light on how the various anchoring groups influence the properties of the dyes. The biscarbodithiolic acid-based dye shows the longest maximum absorption wavelength and the widest absorption spectra together with the highest light-harvesting efficiency, the longest lifetime of the excited state and the highest the LUMO orbital weight of the dye on the atoms of the anchoring group, suggesting the good ability in electron injection. Theoretical calculations have been also performed on the adsorption of these dyes on the TiO₂ anatase (101) surface. These results show that the biscarbodithiolic acid-based dye has the highest adsorption energy and the largest negative shift of the conduction band of TiO₂ due to the adsorption of the dye onto the TiO₂.     

Spectroscopic study of interaction of styrylcyanine dye Sbt and its derivatives with bovine serum albumin

The spectral-fluorescent characteristics of styrylcyanine dye Sbt ((E)-2-(4-(dimethylamino) styryl)-3-methylbenzo[d]thiazol-3-ium iodide) and homodimers, dyes conjugated with two chromophores in aqueous solutions without and in the presence of bovine serum albumin (BSA), are studied. It is established that in the presence of BSA for dyes Dbt-5 and Dbt-10, an increase of the absorptivity, a slight broadening and the emergence of new band on the short wavelength range with λ_max=410 nm is observed; also hypsochromic shift of the absorption and fluorescence at 30 nm and 7 nm, respectively for the dye D-183 is observed. The intensity of the fluorescence emission fundamental band in all the studied dyes in the presence of BSA increases by 3.5 to 55 times. The binding constant (K) and number of binding sites (N) of studied dyes with BSA are determined. The dependence of the binding constants with BSA from the dipole moment of dye molecules is identified, which shows that in addition to the electrostatic attraction forces between molecules of styrylcyanine dyes with BSA, hydrophobic interactions are essential. It is shown that the aggregation of dye affects the processes of interaction of the dyes with the BSA.     

Polymorphism of amyloid-β fibrils and its effects on human erythrocyte catalase binding

The Alzheimer's amyloid-β (Aβ) peptide exists as a number of naturally occurring forms due to differential proteolytic processing of its precursor molecule. Many of the Aβ peptides of different lengths form fibrils in vitro, which often show polymorphisms in the fibril structure. This study presents a TEM based analysis of fibril formation by eighteen different Aβ peptides ranging in length from 5 to 43 amino acids. Spectrophotometric analysis of Congo red binding to the fibrillar material has been assessed and the binding of human erythrocyte catalase (HEC) to Aβ fibrils has also been investigated by TEM. The results show that a diverse range of Aβ peptides form fibrils and also bind Congo red. The ability of both Aβ 1–28 and Aβ 29–40 to form fibrils indicates that there are at least two fibril-forming domains within the full-length Aβ 1–40 sequence, the ability of many Aβ peptides to form Congo red-binding aggregates suggests that there may be up to 4 possible aggregation promoting domains. The binding of HEC was limited to Aβ forms containing residues 29–32. The differing capacities of fibrillar and ribbon-like structures may reflect the accessibility of the 29–32 region and suggest that HEC may be able to discriminate between different forms of Aβ fibrils.     

Two Photon Spectroscopy Can Serve as a Marker of Protein Denaturation Pathway

Rhodamine group of molecules are widely used dyes for imaging of biological molecules. Application of these dyes however includes a limitation that these molecules absorb in the visible range of the spectrum, which does not fall in the ‘biologically transparent window’ (BTW). Two photon absorption (TPA) process could come up with an alternate solution to this as these dyes could be excited in the near infrared (NIR) window to extract similar information. To validate this we have investigated TPA cross section (TPACS, σ₂) of two rhodamine dyes, namely Rhodamine 6G (R6G), Rhodamine B (RhB), site selectively bound with a model protein, bovine serum albumin (BSA), by exciting at 800 nm. Two photon spectroscopy and imaging confirms the binding of the dye to the protein. The decreases in TPACS with increasing temperature at a fixed BSA concentration excellently follows the temperature induced structural transition of BSA as the protein transforms from a molten globule to unfolded conformation beyond 60 °C, which has previously been established through circular dichroism (CD) measurements. The thus established resemblance in TPACS and CD measurement trends thus strongly affirms the suitability of TPA process in protein imaging and as an alternative marker to tracking its conformational transformations using NIR radiation.     

Laser dye stability. Part 5

Photodegradation parameters that relate bleaching and absorption at the lasing wavelength λ_l have been examined for over 30 different coumarin and quinolone laser dyes in a number of solvents. Quinolone dyes were found to bleach faster than the coumarin dyes. The effect of chemical substituents was found to affect bleaching of the coumarin dyes only to a small (20%) extent in ethanol. The major effect of chemical substituents was in the conversion of a dye to products absorbing at λ_l. Effects of solvent, cover gas, and changes in fluorescent quantum yields are discussed. Of particular interest is the photodegradation parameterA, the ratio of the percent absorption at λ_l to the total input energy per dm³. Combined with τ, the total input energy per dm³ required for a laser to reach half its original intensity, it was found thatAτ=1.2±0.9 for all of the dyes independent of dye concentration in all of the solvents tested. It appears that where bleaching of the dye is only of the order of 10–20%, the absorption at λ_l is 1.2% when our dye laser has reached one-half of its initial output. It is consequently possible to estimate τ values of new dyes by the use ofA terms through the relationshipA ₁τ₁=A ₂τ₂ where τ₁ of Dye 1 has been calibrated in the same dye laser system.