muramidase and sodium-sulfate

Addition of high and medium charge density anions (phosphate, sulfate, and chloride) to lysozyme in pure water demonstrates three stages for stabilization of the protein structure. The first two stages have a minor impact on lysozyme stability and are probably associated with direct interaction of the ions with charged and partial charges on the protein's surface. There is a clear transition between the second and third stages; in the case of sodium chloride, disodium sulfate and disodium hydrogen phosphate this is at 550, 210, and 120 mM, respectively. Stabilization of lysozyme can be explained by the free energy required to hydrate the protein as it unfolds. At low ion concentrations, the protein's hydration layer is at equilibrium with the bulk water. After the transition, bulk water is depleted and the protein is competing for water with the ions. With competition for water between the protein and the ions at higher salt concentrations, the free energy required to hydrate the interior of the protein rises and it is this that stabilizes the protein structure.

Topics: Anions; Calorimetry, Differential Scanning; Muramidase; Phosphates; Protein Stability; Protein Unfolding; Sodium Chloride; Sulfates; Temperature; Thermodynamics; Water

Carbon nanotubes (CNTs) were functionalized with sodium hexadecyl sulfate (SHS). The lysozyme adsorbed on the SHS-CNTs exhibited a higher activity than that immobilized on the nonfunctionalized CNTs. To explain the experimental results and explore the mechanism of lysozyme adsorption, large-scale molecular dynamics simulations have been performed for a four-component system, including lysozyme, SHS, CNTs in explicit water. It has been found that the assembled SHS molecules form a soft layer on the surface of CNTs. The interactions between lysozyme and SHS induce the rearrangement of SHS molecules, forming a saddle-like structure on the CNT surface. The saddle-like structure fits the shape of the lysozyme, and the active-site cleft of the lysozyme is exposed to the water phase. Whereas, for the lysozyme adsorbed on the nonfunctionalized CNT, due to the hydrophobic interactions, the active-site cleft of the enzyme tends to face the wall of the CNT. The results of this work demonstrate that the SHS molecules as the interfacial substance have a function of adjusting the lysozyme with an appropriate orientation, which is favorable for the lysozyme having a higher activity.

Topics: Adsorption; Computer Simulation; Entropy; Muramidase; Nanotubes, Carbon; Sulfates; Surface-Active Agents; Water

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.

Topics: Adsorption; Animals; Chickens; Hydrogen-Ion Concentration; Muramidase; Osmolar Concentration; Protein Binding; Silicon Dioxide; Sulfates

The energetics of lysozyme adsorption on aminopropyl-grafted MCF silica (MCF-NH2) are compared to the trends observed during lysozyme adsorption on native MCF silica using flow microcalorimetry (FMC). Surface modification on MCF silica affects adsorption energetics significantly. All thermograms consist of two initial exothermic peaks and one later endothermic peak, but the heat signal trends of MCF-NH2 are opposite from those observed for adsorption onto native MCF silica in salt solutions of sodium acetate and sodium sulfate. At low ionic strength (0.01 M), LYS adsorption onto MCF-NH2 was accompanied by a large exotherm followed by a desorption endotherm. With increasing ionic strength (0.1 and 3.01 M), the magnitude of the thermal signal decreased and the total process became less exothermic. Also a higher protein loading of 14 μmol g(-1) was obtained at low ionic strength in batch adsorption isotherm measurements. Taken together, the FMC thermograms and batch adsorption isotherms reveal that MCF-NH2 has the nature of an ion exchange adsorbent, even though lysozyme and the aminopropyl ligands have like net charges at the adsorption pH. Reduced electrostatic interaction, reduced Debye length, and increased adsorption-site competition attenuate exothermicity at higher ionic strengths. Thermograms from flow microcalorimetry (FMC) give rich insight into the mechanisms of protein adsorption. A two-step adsorption mechanism is proposed in which negatively charged surface amino acid side chains on the lysozyme surface make an initial attachment to surface aminopropyl ligands by electrostatic interaction (low ionic strength) or van der Waals interaction (high ionic strength). Secondary attachments take place between protruding amino acid side chains and silanol groups on the silica surface. The reduced secondary adsorption heat is attributed to the inhibitory effect of the enhanced steric barrier of aminopropyl group on MCF silica.

Topics: Adsorption; Amines; Animals; Calorimetry, Differential Scanning; Chickens; Hydrophobic and Hydrophilic Interactions; Models, Chemical; Muramidase; Osmolar Concentration; Proteins; Silicon Dioxide; Sodium Acetate; Sulfates; Thermodynamics; Thermogravimetry

Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.

Topics: Centrifugation; Chemical Fractionation; Cytochromes c; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Molecular Weight; Muramidase; Plant Extracts; Plant Proteins; Polyethylene Glycols; Ribonuclease, Pancreatic; Sulfates; Water; Zea mays

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45-50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Antibodies, Bacterial; Antibody Formation; Calcium Carbonate; Cells, Cultured; Chromium; Dose-Response Relationship, Drug; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Fish Proteins; Fresh Water; Immunity, Cellular; India; Leukocytes; Muramidase; Reactive Nitrogen Species; Reactive Oxygen Species; Sulfates; Tanning; Tilapia; Time Factors; Water Pollutants, Chemical

The partitioning of model proteins (bovine serum albumin, ovalbumin, trypsin and lysozyme) was assayed in aqueous two-phase systems formed by a salt (potassium phosphate, sodium sulfate and ammonium sulfate) and a mixture of two polyethyleneglycols of different molecular mass. The ratio between the PEG masses in the mixtures was changed in order to obtain different polymer average molecular mass. The effect of polymer molecular mass and polydispersivity on the protein partition coefficient was studied. The relationship between the logarithm of the protein partition coefficient and the average molecular mass of the phase-forming polymer was found to depend on the polyethyleneglycol molecular mass, the salt type in the bottom phase and the molecular weight of the partitioned protein. The polymer polydispersivity proved to be a very useful tool to increase the separation between two proteins having similar isoelectrical point.

Topics: Algorithms; Ammonium Sulfate; Animals; Cattle; Chemical Fractionation; Chemical Phenomena; Chemistry, Physical; Molecular Weight; Muramidase; Ovalbumin; Phosphates; Polyethylene Glycols; Potassium Compounds; Proteins; Reproducibility of Results; Serum Albumin, Bovine; Sulfates; Trypsin

The unfolding of cellular prion protein and its refolding to the scrapie isoform are related to prion diseases. Studies in the literature have shown that structures of proteins, either acidic or basic, are stabilized against denaturation by certain neutral salts, for example, sulfate and fluoride. Contrary to these observations, the full-length recombinant prion protein (amino acid residues 23-231) is denatured by these protein structure stabilizing salts. Under identical concentrations of salts, the structure of the sheep prion protein, which contains a greater number of glycine groups in the N-terminal unstructured segment than the mouse protein, becomes more destabilized. In contrast to the full-length protein, the C-terminal 121-231 prion protein fragment, consisting of all the structural elements of the protein, viz., three alpha-helices and two short beta-strands, is stabilized against denaturation by these salts. We suggest that an increase in the concentration of the anions on the surface of the prion protein molecule due to their preferential interaction with the glycine residues in the N-terminal segment destabilizes the structure of the prion protein by perturbing the prion helix 1 which is the most soluble of all the protein alpha-helices reported so far in the literature. The present results could be relevant to explain the observed structural conversion of the prion protein by anionic nucleic acids and sulfated glycosaminoglycans.

Topics: Amino Acid Sequence; Animals; Buffers; Circular Dichroism; Hot Temperature; Hydrogen-Ion Concentration; Mice; Molecular Sequence Data; Muramidase; Peptide Fragments; Prions; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Salts; Sheep; Solutions; Spectrometry, Fluorescence; Sulfates; Tryptophan

Aqueous two-phase systems have been widely used for the separation and concentration of proteins. In this work we investigated the possibility of using aqueous two-phase system for the renaturation of inclusion body proteins by studying the effect of polyethylene glycol (PEG)-salt systems on the oxidative renaturation of hen egg-white lysozyme (HEWL) with guanidinium chloride (GdmCl) present in the system. To accomplish phase separation at moderately low concentrations of polymer and salt, the total GdmCl concentration had to be kept low (<1 M). The unfolded protein exhibited very low solubility under these conditions. In an attempt to increase the solubility of the protein, temperatures of 40, 50, and 60 degrees C were investigated. The effect of PEG molecular weight was also addressed. Best renaturation yields were obtained when using PEG 3400 and working at 50 degrees C. However, the total protein concentration had to be kept at a low level of 0.2 mg/mL. Lowering the total GdmCl concentration in the system resulted in increased aggregation.

Topics: Animals; Chickens; Eggs; Glycols; Guanidine; Molecular Weight; Muramidase; Polyethylene Glycols; Protein Denaturation; Protein Renaturation; Sulfates; Temperature; Water