muramidase and betadex

Monolayers were formed by specific interactions between adamantylated proteins (transferrin, lysozyme) and a β-cyclodextrin (β-CD) monolayer on a gold surface. Very high stabilities could be reached by multiple interactions of 3-6 adamantyl moieties linked through triethylene glycol spacers to the protein with β-CD rings attached to the surface. Furthermore, bound proteins could be completely removed from the surface through competitive binding of an excess of free adamantane. Regenerable protein sensor chips can be constructed by using this supramolecular toolbox. Attached proteins are still recognized by specific antibodies, which was attributed to a loose packing of the protein molecules at the β-CD monolayer.

Topics: Adamantane; beta-Cyclodextrins; Gold; Humans; Molecular Structure; Muramidase; Surface Plasmon Resonance; Transferrin

An ultrasound-responsive carrier for protein drugs is promising for site-specific release of proteins at disease sites in a designated time course because ultrasound readily penetrates deep into the interior of the body in a non-invasive way. However, the guideline for designing ultrasound-responsive carriers that are applicable to any protein remains to be established. Here, the aim is to develop an ultrasound-responsive material for the controlled release of a variety of proteins regardless of their charge and structure. The supramolecular polymeric hydrogel crosslinked with a host-guest interaction of β-cyclodextrin and adamantane can enclose two kinds of model proteins and site-specifically and stepwisely release them in an ultrasound-guided manner without losing their activities. Furthermore, ultrasound-guided protein delivery to living cells is achieved on model tissue consisting of cells and extracellular matrix. The results of this study provide the proof of principle that the supramolecular polymeric hydrogel is applicable as the core carrier material in an ultrasound-guided protein delivery system.

Topics: beta-Cyclodextrins; Biocompatible Materials; Chemistry Techniques, Synthetic; Drug Delivery Systems; Fluorescence Recovery After Photobleaching; HeLa Cells; Humans; Hydrogels; Muramidase; Polymers; Proteins; Ultrasonic Waves

An electrostatic nanocomplex between naturally occurring ε-poly-l-lysine (εPL) and β-cyclodextrin sulphate (sCD) was designed, and its capacity to entrap four model proteins with high or low molecular weight and isoelectric point, i.e., lactoferrin, albumin, actinidin, and lysozyme, was investigated. The optimal formulations gave nanocomplexes with an average diameter around 276 ± 16 nm, a ζ-potential of -39 ± 1.5 mV, and a spherical shape with a core-shell structure. Different strategies were pursued to increase the entrapment efficiency for selected proteins, which led to 40-100% entrapment depending on the protein type. Under simulated gastric conditions with pepsin, the complexes protected lactoferrin and albumin against proteolysis, whereas actinidin and lysozyme were intrinsically stable. In Caco-2 cells, these complexes transiently decreased the trans-epithelial electrical resistance, indicating the potential to enhance the paracellular permeability of bioactive macromolecules. Thus, these εPL-sCD complexes would be a promising system for loading diverse proteins for gastric protection and enhancing intestinal absorption.

Topics: Albumins; beta-Cyclodextrins; Caco-2 Cells; Cell Survival; Cells, Cultured; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Drug Delivery Systems; Gastrointestinal Tract; Humans; Lactoferrin; Molecular Structure; Muramidase; Particle Size; Polylysine; Protective Agents; Structure-Activity Relationship; Surface Properties

Lysozyme is widely used in medical, food and industrial fields due to its bacteriolytic effect and thus it is significant to design and develop a specific adsorbent with high adsorption capacity and selectivity for lysozyme. Inspired by the high uptake capacity of tetrafluoroterephthalonitrile-crosslinked porous β-cyclodextrin polymers (P-CDPs) and the noncovalent interaction between lysozyme and carboxyl groups, the carboxyl-functionalized P-CDPs (P-CDP-COO

Topics: Adsorption; beta-Cyclodextrins; Carboxylic Acids; Chemistry Techniques, Analytical; Cyclodextrins; Egg White; Muramidase; Porosity

In this work, ionic liquid modified Fe3O4@dopamine/graphene oxide/β-cyclodextrin (ILs-Fe3O4@DA/GO/β-CD) was used as supporting material to synthesize surface molecularly imprinted polymer (SMIP) which then was introduced into chemiluminescence (CL) to achieve an ultrasensitive and selective biosensor for determination of lysozyme (Lys). ILs and β-CD was applied to provide multiple binding sites to prepare Lys SMIP and Fe3O4@DA was designed to make the product separate easily and prevent the aggregation of GO which could improve absorption capacity for its large specific surface area. The ILs-Fe3O4@DA/GO/β-CD-SMIP showed high adsorption capacity (Q = 101 mg/g) to Lys in the adsorption isotherm assays. The adsorption equilibrium was reached within 10 min for all the concentrations, attributing to the binding sites situated exclusively at the surface, and the adsorption model followed Langmuir isotherm. Under the suitable CL conditions, the proposed biosensor could response Lys linearly in the range of 1.0 × 10(-9)-8.0 × 10(-8) mg/mL with a detection limit of 3.0 × 10(-10) mg/mL. When used in practical samples in determination of Lys, the efficient biosensor exhibited excellent result with the recoveries ranging from 94% to 112%.

Topics: beta-Cyclodextrins; Biosensing Techniques; Graphite; Ionic Liquids; Limit of Detection; Luminescence; Magnetics; Microscopy, Electron, Scanning; Molecular Imprinting; Muramidase; Oxides; Surface Properties

The stabilisation of proteins using different excipients in dried forms for possible therapeutic use is extensively studied. However, the effects of excipients on proteins in crystallised forms are sparsely documented. Therefore, the influences of PluronicF-127 and CremophorEL (as surfactants) and β-cyclodextrin and inulin (as sugars) on stability and biological activity of lysozyme, a model protein, in spray dried and crystallised forms were investigated. Spray dried and crystallised lysozyme were prepared in absence and presence of the mentioned excipients in a concentration of 0.05% w/v. The protein formulations were characterised in both solution state (using biological assay, particle size analysis and protein concentration determination) and solid state (employing yield determination, scanning electron microscopic (SEM) examination, Fourier transform infrared (FT-IR) spectroscopy for secondary structure analysis and Differential Scanning Calorimetry (DSC) for thermal study). Also, protein samples were assayed for their biological activities after exposing to storage stability study for 20 weeks in solid states at 24 °C/76% relative humidity (RH) and in aqueous states at 24 °C. The results showed that lysozyme crystals with CremophorEL, PluronicF-127, β-cyclodextrin and inulin maintained protein thermal stability (as indicated by DSC) to greater extent compared with spray dried protein formulations. Also, PluronicF-127 was competent to recover 100% lysozyme from crystallisation protein solutions (as confirmed by yield determination); this surfactant was able to prevent aggregate formation within spray dried lysozyme (as demonstrated by particle size analysis). The presence of PluronicF-127, β-cyclodextrin and inulin preserved the protein biological activity in freshly prepared spray dried and crystallised samples. PluronicF-127 was competent to protect lysozyme in both spray dried and crystallised forms after storage. PluronicF-127 has proved to be a promising protectant of proteins. The improved stability of the spray dried and crystallised protein containing PluronicF-127 shows promise for delivery of proteins via inhalation (in a spray dried form which has particle size range suitable for inhalation as revealed by particle size analysis and SEM) and injectable routes (in spray dried and crystallised forms). The way excipients react with proteins is different in the case of spray drying and crystallisation techniques, hence the choice of the additi

Topics: Acetylglucosamine; beta-Cyclodextrins; Chemistry, Pharmaceutical; Crystallization; Desiccation; Drug Stability; Glycerol; Inulin; Micrococcus; Muramic Acids; Muramidase; Poloxamer; Surface-Active Agents

A novel protein imprinted polymer for selective recognition of lysozyme was obtained. Acryloyl-beta-cyclodextrin, which offered hydrophilic exterior and hydrophobic cavity that were allowed to self-assemble with the template protein through hydrogen interaction and hydrophobic interaction, was synthesized and used as the functional monomer. Polymerization was carried out in the presence of acrylamide as an assistant monomer, which resulted in a new type of protein imprinted polymer. Langmuir adsorption model was employed to describe the isotherms, and maximum adsorption capacity was evaluated. The performance of such imprinted polymer was further demonstrated by high-performance liquid chromatography, and the results showed that the column packed with the lysozyme imprinted silica beads could effectively separate lysozyme from the mixture of lysozyme-cytochrome c, lysozyme-bovine serum albumin, lysozyme-avidin or lysozyme-methylated bovine serum albumin, which showed its high selectivity.

Topics: Acrylamide; Acrylic Resins; Adsorption; Animals; beta-Cyclodextrins; Cattle; Chromatography, High Pressure Liquid; Elements; Microscopy, Electron, Scanning; Microspheres; Molecular Imprinting; Muramidase; Nitrogen; Polymers; Serum Albumin, Bovine; Silicon Dioxide; Solutions; Surface Properties; Temperature

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as beta-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to beta-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.

Topics: beta-Cyclodextrins; Cellulase; Chemical Fractionation; Detergents; Feasibility Studies; Gases; Muramidase; Surface Tension

The interaction of hen egg-white lysozyme with sodium n-dodecyl sulfate (SDS) as an anionic surfactant was investigated by UV-vis spectrophotometry at different pHs at 25 degrees C using HCl/glycine and NaOH/glycine for acidic and basic pH ranges, respectively. Analysis of the spectral data using chemometric method gave the evidence for the existence of intermediate components during the cited interaction. Results also indicated a connection between turbidity of the protein solution upon interaction with SDS and distribution of our newly found intermediates. As intermediates are important in aggregation of proteins, beta-cyclodextrin was employed as an anti-aggregation agent and the results obtained for the lysozyme-SDS-beta-cyclodextrin ternary system were compared with those obtained in the absence of beta-cyclodextrin on distribution and mole fraction of intermediates with. It is also shown that as the distribution of intermediates broadens in a range of SDS concentrations, the turbidity and aggregation state of solution are reduced.

Topics: Animals; beta-Cyclodextrins; Circular Dichroism; Hydrogen-Ion Concentration; Muramidase; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.

Topics: Acrylamide; beta-Cyclodextrins; Biomimetic Materials; Carbonic Anhydrase I; Enzyme Activation; Microspheres; Molecular Chaperones; Muramidase; Phase Transition; Polymers; Protein Folding; Protein Renaturation; Solubility

Carboxymethylated-beta-cyclodextrin (CMBCD) in the electrophoretic medium (aqueous 50 mM sodium phosphate, pH 2.5) enhanced the separation using raw fused-silica capillaries in CZE of the four standard proteins: alpha-chymotrypsinogen A, cytochrome c, lysozyme and ribonuclease A. Furthermore, with 20 mM CMBCD in the electrophoretic medium, the cis-trans isomers of angiotensin could be separated at room temperature, whereas the separation of the conformers required subambient temperatures as low as -20 degrees C without CMBCD in the electrophoretic medium [50 mM sodium phosphate (pH 2.5), containing 10% (v/v) methanol]. Addition of heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) had no effect on the separation of the above proteins and peptides. The results suggest that in microcolumn separation techniques, certain cyclodextrin additives can be useful selectivity enhancers.

Topics: Amino Acid Sequence; Angiotensin I; beta-Cyclodextrins; Chymotrypsinogen; Cold Temperature; Cyclodextrins; Cytochrome c Group; Electrophoresis, Capillary; Muramidase; Proteins; Ribonuclease, Pancreatic

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.

Topics: Adsorption; beta-Cyclodextrins; Chymotrypsinogen; Cyclodextrins; Cytochrome c Group; Electrophoresis, Capillary; Hydrogen-Ion Concentration; Indicators and Reagents; Lactoglobulins; Muramidase; Ovalbumin; Proteins; Ribonuclease, Pancreatic; Sensitivity and Specificity

The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.

Topics: Animals; beta-Cyclodextrins; Carbonic Anhydrases; Cetrimonium; Cetrimonium Compounds; Circular Dichroism; Citrate (si)-Synthase; Cyclodextrins; Detergents; Guanidine; Molecular Chaperones; Muramidase; Myocardium; Protein Conformation; Protein Denaturation; Protein Folding; Swine

A column-switching high-performance liquid chromatography (HPLC) system which consisted of a beta-cyclodextrin (beta-CD) sulfate-immobilized hydrophilic vinyl-polymer gel precolumn and a reversed-phase analytical column was developed for the selective on-line enrichment and separation of heparin-binding proteins. Of 15 proteins investigated, 10 proteins having heparin-binding activity were retained on the beta-CD sulfate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for basic fibroblast growth factor constructed at various sample volumes were nearly identical, indicating that the protein could be enriched by this system. The system was successfully used for the selective separation of lysozyme in egg white. The beta-CD sulfate-immobilized precolumn showed no loss of analytical performance over 2 years during which about 400 samples were analysed.

Topics: beta-Cyclodextrins; Chromatography, High Pressure Liquid; Cyclodextrins; Fibroblast Growth Factor 2; Heparin; Muramidase; Protein Binding; Proteins