muramidase and sodium-nitrate

Metallic biomaterials are widely used to replace and/or restore the function of damaged bodily parts. The use of silver as antibacterial coatings onto implants has recently gained large interest in medical applications. The extent of silver that can be released into different biological fluids from such coatings is, except for the surface characteristics of the coating, governed by parameters such as protein characteristics, adsorbed layer properties, formation of silver-protein complexes as well as concentrations of proteins in the solution. This study aims to relate the structure of adsorbed net negatively charged bovine serum albumin (BSA), which is the most abundant protein in serum, to the release of silver from metallic silver surfaces in order to elucidate if the net charge of the protein has any effect of the silver release. Simultaneous adsorption measurements were performed in real time on the very same surface using combined ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) measurements to provide a more comprehensive understanding on adsorption kinetics and layer structures. The amount of released silver into solution was measured by means of graphite furnace atomic absorption spectroscopy (GF-AAS). The structure of the adsorbed BSA layer largely influenced the amount of released silver, an enhancement that increased with BSA concentration. These observations are in complete contrast to the effect of net positively charged lysozyme (LSZ) adsorbed on silver, previously studied by the authors, for which a complete surface coverage suppressed the possibility for silver release. The underlying mechanisms behind the enhanced release of silver in the presence of BSA were mainly attributed to surface complexation between BSA and silver followed by an enhanced exchange rate of these surface complexes with BSA molecules in the solution, which in turn increase the amount of released silver in solution.

Topics: Adsorption; Animals; Cattle; Graphite; Hydrogen-Ion Concentration; Muramidase; Nitrates; Quartz Crystal Microbalance Techniques; Serum Albumin, Bovine; Silver; Spectrophotometry, Atomic; Surface Properties

Silver is increasingly used in antimicrobial coatings of biomedical devices and implants to hinder infections. As proteins have been shown to largely influence the extent of released metals from various metal surfaces at biological conditions, silver may also be influenced in the same way. The aim of this study is to relate the structure of adsorbed lysozyme (LSZ) to the release of silver from metallic silver surfaces. Simultaneous adsorption measurements were performed in real time on the same surface using combined ellipsometry and quartz crystal microbalance with dissipation monitoring measurements to provide a more comprehensive understanding on the adsorption kinetics and the layer structures. The concentration of LSZ in 0.15 M NaNO3 solution (pH 7, 25 °C) influences the structure of the adsorbed layer. Monolayer coverage is obtained at concentrations ≤0.1 g/L, while a bilayer structure with a rigid inner layer and a relatively loosely adsorbed outer layer is formed at 1 g/L. The inner layer of LSZ is assumed to bind firmly to silver via disulfide bridges, which makes it irreversibly adsorbed with respect to dilution. The amount of released silver is further influenced by the structure of the LSZ layer. At low LSZ concentrations (≤0.1 g/L) the amount of released silver is not significantly different compared with non-protein-containing NaNO3 solutions; however, noticeable reduction was observed at higher concentrations (1 g/L). This reduction in silver release has several possible explanations, including (i) surface complexation between LSZ and silver ions that may result in the incorporation of silver in the irreversible adsorbed layer and, hence, reduce the amount of released silver into solution, and (ii) net charge reversal at the protein/solution interface to slightly positive surface potentials. Any release of silver will therefore exhibit an electrostatic repulsion during transportation through the protein layer results in a reduced amount of silver in solution.

Topics: Adsorption; Muramidase; Nitrates; Quartz Crystal Microbalance Techniques; Silver; Surface Properties

Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T=100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

Topics: Crystallization; Crystallography, X-Ray; Hydroxyl Radical; Muramidase; Nitrates; Protein Conformation; Proteins; Solutions; Temperature

In protein crystallography, spherulites are considered the result of a failed crystallization experiment. Understanding the formation of these structures may contribute to finding methods to prevent their formation. Here, we present an in situ study on lysozyme spherulites grown from sodium nitrate and sodium thiocyanate solutions, investigating their morphology and growth kinetics using optical microscopy. In a morphodrom, we indicate the conditions at which spherulites form for the lysozyme-nitrate system, showing that liquid-liquid phase separation is not a prerequisite to form sheaflike spherulites and that supersaturation is not the only factor determining their creation. Despite their sheaflike morphology, the spherulites all appear to be formed through heterogeneous nucleation. The spherulites are of a new polymorphic form and are less stable than the monoclinic form. For a single needle, growth kinetics indicate surface processes to be the rate-limiting step during growth, but for an entire spherulite volume, diffusion still plays a role. Spherulites simulated by using a time-dependent, tip-splitting model are found to compare well to experimentally observed spherulites.

Topics: Crystallization; Kinetics; Microscopy, Confocal; Muramidase; Nitrates; Protein Conformation; Solutions; Surface Properties; Thiocyanates

Two monoclinic crystals (space group P2(1)) of hen egg-white lysozyme, a type I crystal grown at room temperature in a D2O solution with pD 4.5 containing 2%(w/v) sodium nitrate and a type II crystal grown at 313 K in a 10%(w/v) sodium chloride solution with pH 7.6, were each transformed into another monoclinic crystal with the same space group by dehydration-induced phase transition. Changes in X-ray diffraction were recorded to monitor the progress of the crystal transformation, which started with the appearance of diffuse streaks. In both crystals, the intensity of h + l odd reflections gradually weakened and finally disappeared on completion of the transformation. X-ray diffraction in the intermediate state indicated the presence of lattices of both the native and transformed crystals. In the native type I crystal, two alternate conformations were observed in the main chain of the region Gly71-Asn74. One conformer bound a sodium ion which was replaced with a water molecule in the other conformer. In the transformed crystal, the sodium ion was removed and the main-chain conformation of this region was converted to that of the water-bound form. The transformed crystal diffracted to a higher resolution than the native crystal, while the peak width of the diffraction spots increased. Analysis of the thermal motion of protein molecules using the TLS model has shown that the enhancement of the diffraction power in the transformed crystal is mainly ascribable to the suppression of rigid-body motion owing to an increase in intermolecular contacts as a result of the loss of bulk solvent.

Topics: Animals; Binding Sites; Chickens; Crystallization; Crystallography, X-Ray; Deuterium Oxide; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Muramidase; Nitrates; Phase Transition; Protein Structure, Secondary; Protein Structure, Tertiary; Sodium; Sodium Chloride; Temperature; Water

The ability of acidocin CH5, a bacteriocin from Lactobacillus acidophilus CH5 in the form of neutralized and heated supernatant, to prevent the growth of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 alone or together with other antimicrobials was investigated. The inhibitory activity of acidocin CH5 was higher in MRS broth than in reconstituted skim milk (RSM). In MRS broth and RSM, 1.92 and 32 AU acidocin CH5/ml, respectively, caused 97 and 89% inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of 5 and 10% milk fat in RSM decreased the inhibitory activity of acidocin CH5 to 20 and 11%, respectively. The inhibitory activity of acidocin CH5 was also reduced in the presence of NaCl, NaNO3 and lysozyme. In RSM the inhibition was weaker with both acidocin CH5 and NaCl added compared with NaCl alone. In MRS broth the inhibition was stronger with both acidocin CH5 and NaCl added compared with NaCl alone. The inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was stronger with both NaNO3 and acidocin CH5 in MRS broth (but not in RSM) than with only NaNO3 present, but the strongest level was obtained with acidocin CH5 alone. Addition of acidocin CH5 and more than 30 mg/ml lysozyme to MRS broth increased the level of inhibition above the level obtained by acidocin CH5 alone. The indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was also sensitive to NaCl, NaNO3 and lysozyme in both MRS broth and RSM.

Topics: Animals; Bacteriocins; Colony Count, Microbial; Culture Media; Food Microbiology; Food Preservatives; Lactobacillus; Milk; Muramidase; Nitrates; Sodium Chloride