muramidase and pyrene

A new photochemical reagent, succinic acid-1(1-pyrene)methylamide (PMA-SUC), was developed to recognize the specific binding sites on model proteins, egg-white lysozyme and avidin. The interaction of the photochemical reagent with the proteins was studied by UV-Vis, fluorescence spectroscopic methods and docking description. PMA-SUC was found to bind to lysozyme and avidin with binding constants (K

Topics: Animals; Avidin; Binding Sites; Chickens; Hydrogen Bonding; Molecular Docking Simulation; Muramidase; Photolysis; Protein Binding; Protein Structure, Tertiary; Pyrenes; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Succinates; Ultraviolet Rays

The fabrication of a versatile nanocarrier based on agglomerated structures of gold nanoparticle (Au NP)-lysozyme (Lyz) in aqueous medium is reported. The carriers exhibit efficient loading capacities for both hydrophilic (doxorubicin) and hydrophobic (pyrene) molecules. The nanocarriers are finally coated with an albumin layer to render them stable and also facilitate their uptake by cancer cells. The interaction between agglomerated structures and the payloads is non-covalent. Cell viability assay in vitro showed that the nanocarriers by themselves are non-cytotoxic, whereas the doxorubicin-loaded ones are cytotoxic, with efficiencies higher than that of the free drug. Transmission electron microscopy and fluorescence microscopy along with flow cytometry analysis confirm the uptake of the drug-loaded nanocarriers by a human cervical cancer HeLa cell line. Field-emission scanning electron microscopy reveals the formation of apoptotic bodies leading to cell death, confirming the release of the payloads from the nanocarriers into the cell. Overall, the findings suggest the fabrication of novel Au NP-protein agglomerate-based nanocarriers with efficient drug-loading and -releasing capabilities, enabling them to act as multimodal drug-delivery vehicles.

Topics: Animals; Cell Survival; Chickens; Doxorubicin; Drug Carriers; Drug Delivery Systems; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Gold; HeLa Cells; Humans; Metal Nanoparticles; Muramidase; Pyrenes

The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.

Topics: 2-Naphthylamine; Amyloid; Animals; Benz(a)Anthracenes; Cell Membrane; Diphenylhexatriene; Fluorescent Dyes; Laurates; Lipid Bilayers; Muramidase; Protein Multimerization; Protein Structure, Secondary; Pyrenes; Spectrometry, Fluorescence; Water

The ability of oligomeric lysozyme to modify the molecular organization of the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with phosphatidylglycerol (PG) or cholesterol (Chol) was assessed using fluorescent probes 6-propionyl-2-dimethylaminonaphthalene (Prodan), 4-dimethylaminochalcone (DMC), pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH). The observed changes in the fluorescence characteristics of polarity-sensitive probes Prodan and DMC, located in interfacial bilayer region, were interpreted due to the partial dehydration of the glycerol backbone, which was under the influence of aggregated protein. Cholesterol was found to prevent the perturbations of membrane polar part by lysozyme aggregates. Analysis of the pyrene excimerization data revealed an oligomer-induced reduction in bilayer free volume, presumably caused by an increased packing density of hydrocarbon chains. This effect proved to be virtually independent of membrane composition. It was demonstrated that membranotropic activity of oligomeric lysozyme markedly exceeds that of monomeric protein. The biological significance of the results obtained is twofold, implicating the general membrane-mediated mechanisms of oligomer toxicity and specific pathways of lysozyme fibrillogenesis in vivo associated with familial nonneuropathic systemic amyloidosis.

Topics: 2-Naphthylamine; Chalcones; Cholesterol; Diphenylhexatriene; Fluorescent Dyes; Lipid Bilayers; Models, Molecular; Muramidase; Phosphatidylcholines; Polymers; Pyrenes; Spectrometry, Fluorescence

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein-protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A-H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A-H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A-H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A-H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.

Topics: Actin Cytoskeleton; Actins; Animals; Chickens; Histones; Microscopy, Atomic Force; Muramidase; Peptide Elongation Factor 1; Polymerization; Protein Binding; Protein Conformation; Pyrenes; Rabbits; Saccharomyces cerevisiae Proteins; Static Electricity

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification and prior sample purification. To address these limitations, we report here the design, synthesis, and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by a steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), showing the generality of our approach. Therefore, the competition-mediated pyrene-switching aptasensor is promising to have potential for clinical and forensic applications.

Topics: Aptamers, Nucleotide; Binding, Competitive; Fluorescence; Humans; Muramidase; Patient Acceptance of Health Care; Pyrenes

Steady-state measurements of pyrene fluorescence in the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) have been performed to gain insight into the effect of lysozyme on molecular organization of lipid bilayer. Analysis of vibronic structure of the probe emission spectra revealed no changes in transverse distribution of pyrene monomers on varying CL contents or increasing the extent of lysozyme binding to liposomes. Excimer-to-monomer fluorescence intensity ratio has been found to reduce on lysozyme association with lipids. The magnitude of this effect increased with increasing CL content from 0 to 40 mol%. These results have been interpreted as indicating decrease in the membrane free volume on formation of both electrostatic and hydrophobic protein-lipid contacts.

Topics: Cardiolipins; Membrane Lipids; Membranes, Artificial; Muramidase; Phosphatidylcholines; Pyrenes