muramidase and triphosphoric-acid

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.

Topics: Adenosine; Adenosine Triphosphate; Binding Sites; Muramidase; Polyphosphates; Protein Binding

The mechanism by which sodium tripolyphosphate affected the aggregation behavior of ovalbumin-lysozyme complexes was investigated in this work. The highest stability coefficients were detected for natural ovalbumin and lysozyme at pH 9.0 and pH 5.0, with values of 0.981 and 0.931, respectively. The turbidity of the phosphorylated ovalbumin-lysozyme complexes was 1.71-fold to the natural complexes at pH 7.0. This result was related to the fact that the phosphorylated sample had a lower isoelectric point. Besides, both intermolecular forces and SDS-PAGE analysis indicated that the disulfide bond was the most important interaction in the complex. Circular dichroism analysis showed that phosphorylation weakened the unfolding and stretching of the structure caused by heat treatment. Moreover, transmission electron microscopy pictures confirmed that the network structure of phosphorylated ovalbumin-lysozyme complex was broader than natural protein. This study provides information for further understanding the effect of phosphorylation on protein aggregation behavior.

Topics: Hydrogen-Ion Concentration; Muramidase; Ovalbumin; Phosphorylation; Polyphosphates; Protein Aggregates; Protein Binding

To understand the effect of counter ions (Na

Topics: Animals; Catalytic Domain; Chickens; Chitosan; Cross-Linking Reagents; Ferric Compounds; Hydrogen-Ion Concentration; Metal Nanoparticles; Micrococcus; Molecular Weight; Muramidase; Polyphosphates; Protein Binding; Protein Structure, Secondary; Sodium; Static Electricity

The ability of polyvalent anions to influence protein-protein interactions and protein net charge was investigated through solubility and turbidity experiments, determination of osmotic second virial coefficients ( B

Topics: Chemical Precipitation; Chlorides; Citric Acid; Diphosphates; Muramidase; Polyphosphates; Protein Binding; Protein Multimerization; Scattering, Radiation; Sulfates

Chitosan/carrageenan/tripolyphosphate nanoparticles were previously presented as holding potential for an application in transmucosal delivery of macromolecules, with tripolyphosphate demonstrating to contribute for both size reduction and stabilisation of the nanoparticles. This work was aimed at evaluating the capacity of the nanoparticles as protein carriers for pulmonary and nasal transmucosal delivery, further assessing their biocompatibility pattern regarding that application. Nanoparticles demonstrated stability in presence of lysozyme, while freeze-drying was shown to preserve their characteristics when glucose or sucrose were used as cryoprotectants. Bovine serum albumin was associated to the nanoparticles, which were successfully microencapsulated by spray-drying to meet the aerodynamic requirements inherent to pulmonary delivery. Finally, a satisfactory biocompatibility profile was demonstrated upon exposure of two respiratory cell lines (Calu-3 and A549 cells) to the carriers. A negligible effect on cell viability along with no alterations on transepithelial electrical resistance and no induction of inflammatory response were observed.

Topics: Biocompatible Materials; Carrageenan; Cell Line; Cell Survival; Chitosan; Cryoprotective Agents; Drug Carriers; Drug Compounding; Freeze Drying; Humans; Monosaccharides; Muramidase; Nanoparticles; Polymers; Polyphosphates; Serum Albumin, Bovine