muramidase and acetonitrile

Preferential solvation/hydration is an effective way for regulating the mechanism of the protein destabilization/stabilization. Organic solvent/water sorption and residual enzyme activity measurements were performed to monitor the preferential solvation/hydration of hen egg-white lysozyme at high and low water content in acetonitrile at 25 °C. The obtained results show that the protein destabilization/stabilization depends essentially on the initial hydration level of lysozyme and the water content in acetonitrile. There are three composition regimes for the dried lysozyme. At high water content, the lysozyme has a higher affinity for water than for acetonitrile. The residual enzyme activity values are close to 100%. At the intermediate water content, the dehydrated lysozyme has a higher affinity for acetonitrile than for water. A minimum on the residual enzyme activity curve was observed in this concentration range. At the lowest water content, the organic solvent molecules are preferentially excluded from the dried lysozyme, resulting in the preferential hydration. The residual catalytic activity is ∼80%, compared with that observed after incubation in pure water. Two distinct schemes are operative for the hydrated lysozyme. At high and intermediate water content, lysozyme is preferentially hydrated. However, in contrast to the dried protein, at the intermediate water content, the initially hydrated lysozyme has the increased preferential hydration parameters. At low water content, the preferential binding of the acetonitrile molecules to the initially hydrated lysozyme was detected. No residual enzyme activity was observed in the water-poor acetonitrile. Our data clearly show that the initial hydration level of the protein macromolecules is one of the key factors that govern the stability of the protein-water-organic solvent systems.

Topics: Acetonitriles; Muramidase; Solubility; Water

A type of mixed-mode chromatography was integrated with high-performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed-mode column, the silica beads were activated with γ-(2,3-epoxypropoxy)-propytrimethoxysilane and coupled with 4-mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.

Topics: Acetonitriles; Animals; Cattle; Chromatography, High Pressure Liquid; Hydrogen-Ion Concentration; Models, Molecular; Muramidase; Serum Albumin, Bovine; Silicon Dioxide; Sodium Chloride

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Topics: 2-Propanol; Acetone; Acetonitriles; Acetylcholinesterase; Animals; Chickens; Chitinases; Chymotrypsin; Enzyme Assays; Ethanol; Humans; Methanol; Muramidase; Neutrophils; Pancreas; Pancreatic Elastase; Solvents; Spectrometry, Mass, Electrospray Ionization; Swine; Time Factors

We describe the preparation of a capillary trypsin immobilized monolithic enzyme reactor (IMER) for a rapid and efficient digestion of proteins down to the femtomole level. Trypsin was immobilized on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith using the glutaraldehyde technique. Digestion efficiencies of the IMER were evaluated using model proteins and protein mixtures as well as chemically cross-linked lysozyme regarding the addition of denaturants and increasing digestion temperature. The trypsin IMER described herein is applicable for the digestion of protein mixtures. Even at a 1000-fold molar excess of one protein, low-abundance proteins are readily identified, in combination with MS/MS analysis. An online setup of the IMER with reversed phase nano-HPLC separation and nano-ESI-MS/MS analysis was established. The great potential of the trypsin IMER for proteomics applications comprise short digestion times in the range of seconds to minutes, in addition to improved digestion efficiencies, compared to in-solution digestion.

Topics: Acetonitriles; Analytic Sample Preparation Methods; Animals; Cattle; Cytochromes c; Enzymes, Immobilized; Guanidine; Humans; Muramidase; Online Systems; Polymers; Protein Denaturation; Proteins; Serum Albumin, Bovine; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Urea

A neutral hydroxylated octadecyl monolith (ODM-OH) for reversed-phase capillary electrochromatography has been developed. The ODM-OH was prepared by the in situ polymerization of octadecyl acrylate and pentaerythritol triacrylate (PETA) in a ternary porogenic solvent. Pentaerythritol triacrylate possesses a hydroxyl functional group, which imparts the monolith with a hydrophilic group, thus the acronym ODM-OH. The ODM-OH column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. This ODM-OH monolith exhibited stronger EOF than its counterpart the ODM made from the in situ polymerization of octadecyl acrylate and trimethylolpropane trimethacrylate. Similar to ODM, it is believed that the EOF was due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting the neutral monolithic column the zeta potential necessary to support the EOF. The higher EOF exhibited by ODM-OH was due to the presence of polar OH groups on its surface, which would favor stronger adsorption of ions from the mobile phase. The wide applications of the neutral ODM-OH column were demonstrated in the separation of a wide range of small and large solutes. As a typical result, the ODM-OH was able to separate proteins quite rapidly yielding 200,000 plates/m.

Topics: Acetonitriles; Acrylates; Capillary Electrochromatography; Electroosmosis; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Muramidase; Peptide Fragments; Peptide Mapping; Polycyclic Aromatic Hydrocarbons; Polymerization; Propylene Glycols; Static Electricity; Trypsin

Crystallizing solutions of proteins often contain various nonelectrolyte additives that arise from the purification process of proteins or from the reagents employed in the screening kits. Currently, limited knowledge exists about the influence of these additives on the mechanisms underlying the crystallization process, in particular on the nucleation stage of crystals. To address this need, we studied crystallization of two proteins, D-xylose isomerase and chicken egg-white lysozyme, in small batches and in the presence of two solubility-enhancing additives, acetonitrile and glycerol. We have also measured the nucleation rates of crystals of these proteins in the presence and in the absence of acetonitrile using the method of initial rates. With the addition of the solubility enhancers, both proteins exhibited an increase in crystal nucleation at any given supersaturation. Solubility enhancing additives appear to lower the energy barrier to nucleation by influencing the strength of attraction between the protein molecules. We have characterized the quality of D-xylose isomerase crystals by determining the crystal mosaicity, which showed considerable improvement for crystals grown in the presence of additives. When compared to the crystals of chicken egg-white lysozyme, D-xylose isomerase crystals required higher supersaturations to nucleate. We attribute this result to the large size of the D-xylose isomerase molecule, which influences the energy barrier to nucleation by increasing the surface area of the critical nucleus. Contrary to the common expectation that reagents that solubilize the protein may hinder the crystallization process, our results suggest that solubility enhancers, in fact, can have a beneficial effect on the nucleation and growth of crystals. These findings are of importance in formulating successful strategies toward crystallizing new proteins.

Topics: Acetonitriles; Animals; Biophysics; Cell Proliferation; Chickens; Crystallization; Egg White; Kinetics; Models, Statistical; Muramidase; Proteins; Solubility; Streptomyces; Temperature; Xylose

Photoprocesses of 1,4-naphthoquinone (NQ) and its photoreactions with lysozyme in acetonitrile/water (3:1, v/v) solution were studied using 355 nm laser flash photolysis technique combined with electrophoresis and turbidimetric assay. The transient spectra of NQ were observed and the transient species were assigned. The electron transfer process from N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to NQ triplet state ((3)NQ) was investigated and the rate constant was determined to be k(t1)=2.0 x 10(10)M(-1)s(-1). It has been found that (3)NQ can abstract hydrogen atom from lysozyme with a rate constant of k(t2)=2.4 x10(10)M(-1)s(-1). Furthermore, the results of steady-state analysis suggested that lysozyme can be damaged by NQ irradiated with UVA light influenced by the concentration of NQ and the gas saturated in the solution. The mechanisms of photosensitized damage of lysozyme were discussed.

Topics: Acetonitriles; Kinetics; Lasers; Muramidase; Naphthoquinones; Photolysis; Photosensitivity Disorders; Tetramethylphenylenediamine; Water

Computational mapping methods place molecular probes (small molecules or functional groups) on a protein surface to identify the most favorable binding positions by calculating an interaction potential. We have developed a novel computational mapping program called CS-Map (computational solvent mapping of proteins), which differs from earlier mapping methods in three respects: (i) it initially moves the ligands on the protein surface toward regions with favorable electrostatics and desolvation, (ii) the final scoring potential accounts for desolvation, and (iii) the docked ligand positions are clustered, and the clusters are ranked on the basis of their average free energies. To understand the relative importance of these factors, we developed alternative algorithms that use the DOCK and GRAMM programs for the initial search. Because of the availability of experimental solvent mapping data, lysozyme and thermolysin are considered as test proteins. Both DOCK and GRAMM speed up the initial search, and the combined algorithms yield acceptable mapping results. However, the DOCK-based approaches place the consensus site farther from its experimentally determined position than CS-Map, primarily because of the lack of a solvation term in the initial search. The GRAMM-based program also finds the correct consensus site for thermolysin. We conclude that good sampling is the most important requirement for successful mapping, but accounting for desolvation and clustering of ligand positions also help to reduce the number of false positives.

Topics: 2-Propanol; Acetone; Acetonitriles; Algorithms; Binding Sites; Binding, Competitive; Computer Simulation; Egg Proteins; Models, Molecular; Muramidase; Phenol; Protein Binding; Proteins; Solvents; Thermodynamics; Thermolysin

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.

Topics: Acetonitriles; Animals; Calorimetry, Differential Scanning; Chickens; Enzyme Stability; Hydrogen-Ion Concentration; Muramidase; Protein Denaturation; Solutions; Temperature; Thermodynamics; Water

Topics: Acetonitriles; Animals; Antibody Formation; Antigen-Presenting Cells; Cell Survival; Corticosterone; Hypersensitivity, Delayed; Immunity, Cellular; Immunity, Innate; Lethal Dose 50; Lysine; Macrophages, Peritoneal; Muramidase; Neutrophils; Poisoning; Rats; Rats, Wistar; Spleen

Tetragonal crystals of hen egg white lysozyme were cross-linked and subjected to X-ray diffraction study in acetonitrile-water media with different acetonitrile concentrations. Crystals in neat acetonitrile did not scatter X-ray well. Structures of crystals in neat water, in 90% and 95% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% and 95% acetonitrile, only one protein-bond acetonitrile molecule is found in the active site cleft, and its location and binding-protein mode is similar to the C subunit of polysaccharide. The alteration in conformation and hydrogen-bond pattern involving water as solvent causes the reduction of the protein's flexibility in organic media. The back-soaked crystal regained its ordinary three-dimensional structure in water.

Topics: Acetonitriles; Cross-Linking Reagents; Crystallography, X-Ray; Models, Molecular; Muramidase; Protein Conformation

The solubility of amino acids and the preferential solvent interaction of hen-egg lysozyme in acetonitrile (AN)-water mixtures (<60 w/v% AN) were investigated by means of densimetric and refractometric methods at 25 degreesC. The free energy of transfer from water to aqueous AN was negative for most nonpolar side-chains of amino acids and positive for the peptide group, the extent being comparable to those for methanol and ethanol systems. Addition of AN to an aqueous solvent was thus suggested to weaken the hydrophobic interaction and to enhance the peptide-peptide hydrogen bond therein leading to the denaturation of proteins. A parallel examination by circular dichroism confirmed that the conformation of lysozyme (pH 3) remains native in aqueous AN up to 40% but changes to the helix-rich form at higher AN concentrations. At all solvent compositions up to 50% AN (pH 3), however, lysozyme was preferentially hydrated probably due to a local salting-out of the AN molecules from the charges on the protein surface, indicating the increase of the chemical potential of the protein. These results are discussed in relation to the role of AN as an eluting organic solvent in reverse-phase chromatography.

Topics: Acetonitriles; Amino Acids; Animals; Chickens; Circular Dichroism; Densitometry; Ethanol; Hydrogen Bonding; Methanol; Muramidase; Protein Denaturation; Protein Structure, Secondary; Refractometry; Solubility; Solvents; Thermodynamics

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses.. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis.. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA.. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

Topics: Acetonitriles; Chromatography, High Pressure Liquid; Contact Lens Solutions; Contact Lenses, Hydrophilic; Drug Combinations; Eye Proteins; Humans; Methods; Muramidase; Quinolines; Solvents; Surface Properties; Tears; Trifluoroacetic Acid

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Topics: Acetates; Acetic Acid; Acetonitriles; Chromatography, High Pressure Liquid; Diabetes Mellitus; Humans; Insulin; Lactoglobulins; Muramidase; Ovalbumin; Pancreas; Peptides; Polymers; Serum Albumin; Trifluoroacetic Acid

A procedure is described for the removal of sodium dodecyl sulphate (SDS) from proteins isolated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated by SDS-PAGE were stained with Coomassie Blue and extracted with Tris-HCl buffer containing SDS. The obtained extracts were subjected to gel permeation chromatography in an acidic aqueous acetonitrile solution. The procedure allows purification of the isolated proteins not only from SDS, but also from Coomassie Blue, buffer salts and other small molecular weight contaminants.

Topics: Acetonitriles; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Lactoglobulins; Muramidase; Ovalbumin; Proteins; Serum Albumin, Bovine; Serum Amyloid A Protein; Sodium Dodecyl Sulfate; Trifluoroacetic Acid