muramidase and nile-red

Many proteins can abnormally fold to form pathological amyloid deposits/aggregates that are responsible for various degenerative disorders called amyloidosis. Here we have examined the anti-amyloidogenic potency of an ionic liquid, 1-butyl-3-methylimidazolium bromide, using lysozyme as a model system. Thioflavin T fluorescence assay demonstrated that the ionic liquid suppressed the formation of lysozyme fibrils significantly. This observation was further confirmed by the Congo red assay. Fluorescence microscopy, intrinsic fluorescence studies, nile red fluorescence assay, ANS binding assay and circular dichroism studies also testified diminishing of the fibrillogenesis in the presence of ionic liquid. Formation of amyloid fibrils was also characterized by α to β conformational transition. From far-UV circular dichroism studies it was observed that the β-sheet content of the lysozyme samples decreased in the presence of the ionic liquid which in turn implied that fibrillogenesis was supressed by the ionic liquid. Atomic force microscopy imaging unequivocally established that the ionic liquid attenuated fibrillogenesis in lysozyme. These results may be useful for the development of more effective therapeutics for amyloidosis.

Topics: Amyloid; Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Chickens; Circular Dichroism; Congo Red; Imaging, Three-Dimensional; Imidazoles; Ionic Liquids; Light; Microscopy, Atomic Force; Muramidase; Oxazines; Spectrometry, Fluorescence; Thiazoles

Polyglycidol-based nanohydrogels (nHGs) have been prepared by optimizing the use of liposome master templates resulting in a high-yielding and more practical one-pot process to provide materials capable of carrying drugs of adverse chemical nature. The nanogels prepared with the one-pot method showed favorable kinetics for the release of either Nile Red (NR) or lysozyme (LYS), loaded with gel precursors such as semi-branched poly(glycidol allylglycidyl ether), PEG dithiol (1KDa), a free radical initiator and liposomal lipids at the liposome formation step. This process is superior to a comparable step-wise traditional approach and circumvents loading of the gel precursors with the hydrophilic drug into preformed liposome templates. A thiol-ene crosslinking reaction accomplishes the formation of the nanonetwork resulting in nHGs prepared in the traditional step-wise (nHG-SW) approach and the one-pot (nHG-OP) process. Both nanogel networks were characterized in terms of particle size and zeta (ζ) potential with average values of 148nm±39nm and -25.9mV±9.2 for the nHG-SW and 132nm±32 and -23.1mV±9.7 for the nHG-OPs. Loading efficiency for both of the nanogels with NR was determined by spectrophotometry to be 28% (nHP-SW) and 31% (nHP-OP). The LYS loading was based on the target loading of 10μg/mg for both nanogels found to be 84% and 86% for the nHG-SW and nHP-OP, respectively. As proof of concept for combination drug delivery, the in vitro release of both drug mimics, NR and LYS, were monitored under physiologically relevant conditions by an optimized dialysis method. The implementation of the multi-functional and semi-branched polyglycidol is recognized as the main contributor for the observed highly controlled release of proteins that are otherwise rapidly released from common PEG-based nanogel networks. Furthermore, the one-pot process led to be the most favorable drug delivery system based on the release kinetics pointing to a denser polymer network.

Topics: Drug Delivery Systems; Drug Liberation; Hydrophobic and Hydrophilic Interactions; Liposomes; Muramidase; Nanoparticles; Oxazines; Particle Size; Propylene Glycols

Polymer nanoparticles are prepared by self-assembly of visible light and pH sensitive perylene-functionalized copolymers which are synthesized by quaternization between 1-(bromomethyl)perylene and the dimethylaminoethyl units of poly(dimethylaminoethyl methacrylate) (PDMAEMA). The perylene-containing polymethacrylate segments afford the system visible light responsiveness and the unquaternized PDMAEMA segments afford the system pH responsiveness. The self-assembled nanoparticles exhibit a unique dual stimuli response. They can be photocleaved under visible light irradiation, shrunken to smaller nanoparticles at high pH, and swollen at low pH. The structural change endows the nanoparticle with great potential as a sensitive nanocarrier for controlled release of Nile Red and lysozyme under this stimulation. The visible light responsiveness and synergistic effect on the release of loaded molecules with the dual stimulation may obviate the need for harsh conditions such as UV light or extreme pH stimulation, rendering the system more applicable under mild conditions.

Topics: Cell Membrane; Delayed-Action Preparations; Drug Carriers; Hydrogen-Ion Concentration; Light; Methacrylates; Micrococcus; Muramidase; Nanoparticles; Nylons; Oxazines; Polymers

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting Aβ1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of Aβ1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.

Topics: Amyloid; Benzothiazoles; Binding Sites; Fluorescence Resonance Energy Transfer; Humans; Hydrogen-Ion Concentration; Kinetics; Microscopy; Muramidase; Oxazines; Prealbumin; Protein Binding; Staining and Labeling; Thiazoles

Liquid-encapsulated lysozyme microspheres were successfully synthesized using a sonochemical method. The encapsulation of four different liquids, namely, sunflower oil, tetradecane, dodecane and perfluorohexane on the formation, stability and morphology of the lysozyme microspheres was studied. Among the four different liquids used for encapsulation, perfluorohexane-filled microspheres were found to be most stable in the dried state with a narrow size distribution. In order to explore the possibility of encapsulating biofunctional molecules (e.g., drugs) within these microspheres, liquids containing a fluorescent dye (Nile red) were encapsulated and the ultrasound-induced release of these dye-loaded liquids was studied. The fluorescence data for the liquid-filled lysozyme microspheres demonstrated the potential use of the sonochemical technique for synthesizing these "vehicles" for the encapsulation and the controlled delivery of dyes, flavours, fragrances or drugs.

Topics: Acoustics; Alkanes; Animals; Cattle; Chemistry; Chickens; Fluorescent Dyes; Fluorocarbons; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Muramidase; Oxazines; Plant Oils; Proteins; Sunflower Oil; Ultrasonics

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.

Topics: Amino Acid Sequence; Amyloid; Animals; Chickens; Chromatography, Gel; Fluorescence; Humans; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Muramidase; Oxazines; Peptide Fragments; Protein Binding; Protein Denaturation; Protein Structure, Quaternary; Protein Structure, Secondary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Thermodynamics; Time Factors

A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.

Topics: Azo Compounds; Light; Muramidase; Oxazines; Photochemistry; Photosensitizing Agents; Protein Conformation; Protein Folding; Quaternary Ammonium Compounds; Scattering, Radiation; Spectrometry, Fluorescence; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Surface-Active Agents

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.

Topics: Catalase; Fluorescent Dyes; Histones; Lactoglobulins; Micelles; Muramidase; Oxazines; Proteins; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Trypsinogen; X-Ray Diffraction