muramidase and alpha-chymotrypsin

A review in which many original published results of the authors as well as many other papers are discussed. The structure and some properties of the globular proteins are shortly presented, special accent being put on the alpha-chymotrypsin (alpha-ChT), lysozyme (LZ), human serum albumin (HSA), and bovine serum albumin (BSA) which have been used in the experiments with thin liquid films. The behaviour of protein adsorption layers (PAL) is extensively discussed. The dynamics of PAL formation, including the kinetics of adsorption as well as the time evolution of the surface tension of protein aqueous solutions, are considered. A considerable place is devoted to the surface tension and adsorption isotherms of the globular protein solutions, the simulation of PAL by interacting hard spheres, the experimental surface tension isotherms of the above mentioned proteins, and the interfacial tension isotherms for the protein aqueous solution/oil interface. The rheological properties of PAL at fluid interfaces are shortly reviewed. After a brief information about the experimental methods for investigation of protein thin liquid (foam or emulsion) films, the properties of the protein black foam films are extensively discussed: the conditions for their formation, the influence of the electrolytes and pH on the film type and stability, the thermodynamic properties of the black foam films, the contact angles film/bulk and their dynamic hysteresis. The next center of attention concerns some properties of the protein emulsion films: the conditions for formation of emulsion black films, the formation and development of a dimpling in microscopic, circular films. The protein-phospholipid mixed foam films are also briefly considered.

Topics: Adsorption; Chymotrypsin; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Molecular Conformation; Muramidase; Phospholipids; Protein Conformation; Protein Structure, Secondary; Proteins; Rheology; Serum Albumin; Serum Albumin, Bovine

The interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin and lysozyme was investigated using multispectral and molecular docking methods. The results of fluorescence quenching revealed that myricetin and dihydromyricetin could quench the intrinsic fluorescence of three different proteinases through a static quenching procedure. The binding constant and number of binding sites at different temperatures were measured. The thermodynamic parameters obtained at different temperatures showed van der Waals interactions and hydrogen bonds played the main roles in the interaction of myricetin with trypsin and lysozyme, hydrophobic force was dominant both in myricetin with α-chymotrypsin interaction and dihydromyricetin with trypsin and lysozyme interaction, as for the electrostatic forces, it was mainly the driving force in dihydromyricetin binding to α-chymotrypsin. There was non-radiative energy transfer between three proteinases and myricetin or dihydromyricetin with high probability. The microenvironment of trypsin, α-chymotrypsin and lysozyme is changed. The docking studies revealed that myricetin and dihydromyricetin entered the hydrophobic cavity of three proteinases and formed hydrogen bonds. The binding affinity of myricetin or dihydromyricetin is different with the trypsin, α-chymotrypsin and lysozyme due to the different molecular structure.

Topics: Binding Sites; Chymotrypsin; Flavonoids; Flavonols; Molecular Docking Simulation; Muramidase; Protein Binding; Spectrometry, Fluorescence; Thermodynamics; Trypsin

Multi-component adsorption of proteins still requires a better understanding of local phenomena to improve the development of predictive models. In this work, all-atom Molecular Dynamics (MD) simulations were used to investigate the influence of protein charge distribution on the adsorption capacity. The simultaneous adsorption of α-chymotrypsin and lysozyme on a cation exchanger, SP Sepharose FF, was studied through MD simulations and compared to macroscopic isotherm experiments. It appears that the charge distribution is a relevant information to better understand specific phenomena, such as a multilayer adsorption caused by the particular electrostatic profile of α-chymotrypsin. Therefore, MD simulations seem to be an interesting way to visualize and highlight these behaviors.

Topics: Adsorption; Chromatography, Ion Exchange; Chymotrypsin; Molecular Dynamics Simulation; Muramidase; Surface Properties

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO

Topics: Animals; Cattle; Chickens; Chromatography, Liquid; Chymotrypsin; Chymotrypsinogen; Mass Spectrometry; Muramidase; Plant Proteins; Ribonuclease, Pancreatic

Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer ("soapy shell") of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation.

Topics: Animals; Buffers; Cattle; Chickens; Chymotrypsin; Circular Dichroism; Glycerol; Hexanes; Immobilized Proteins; Muramidase; Ovalbumin; Polidocanol; Polyethylene Glycols; Protein Conformation; Refractometry; Serum Albumin, Bovine; Solutions; Solvents

Conjugates of enzymes and poly(2-methyloxazoline) were used as organosoluble amphiphilic polymer nanocontainers for dissolving osmate, thereby converting the enzymes into organosoluble artificial metalloenzymes. These were shown to catalyze the dihydroxylation of different alkenes with high enantioselectivity. The highest selectivities, found for osmate complexed with laccase polymer-enzyme conjugates (PECs), even exceed those of classical Sharpless catalysts.

Topics: Alkenes; Biocatalysis; Chymotrypsin; Fungal Proteins; Hydroxylation; Laccase; Ligands; Lipase; Muramidase; Oxazoles; Peroxidase; Polymers; Solvents; Stereoisomerism

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to β-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function.

Topics: Animals; Catalase; Cattle; Chickens; Chymotrypsin; Desiccation; Enzyme Activation; Freeze Drying; Glass; Horseradish Peroxidase; Microtechnology; Muramidase

Abstract Poly(glycerol adipate-co-ω-pentadecalactone) (PGA-co-PDL) was previously evaluated for the colloidal delivery of α-chymotrypsin. In this article, the effect of varying polymer molecular weight (MW) and chemistry on particle size and morphology; encapsulation efficiency; in vitro release; and the biological activity of α-chymotrypsin (α-CH) and lysozyme (LS) were investigated. Microparticles were prepared using emulsion solvent evaporation and evaluated by various methods. Altering the MW or monomer ratio of PGA-co-PDL did not significantly affect the encapsulation efficiency and overall poly(1,3-propanediol adipate-co-ω-pentadecalactone) (PPA-co-PDL) demonstrated the highest encapsulation efficiency. In vitro release varied between polymers, and the burst release for α-CH-loaded microparticles was lower when a higher MW PGA-co-PDL or more hydrophobic PPA-co-PDL was used. The results suggest that, although these co-polyesters could be useful for protein delivery, little difference was observed between the different PGA-co-PDL polymers and PPA-co-PDL generally provided a higher encapsulation and slower release of enzyme than the other polymers tested.

Topics: Chymotrypsin; Drug Delivery Systems; Emulsions; Lactones; Macrolides; Microspheres; Molecular Weight; Muramidase; Particle Size; Polyesters; Polymers; Propylene Glycols; Proteins

The interface between nematic liquid crystal, 4-cyano-4'-pentylbiphenyl (5CB), and water in a transmission electron microscopy (TEM) grid cell coated with QP4VP-b-LCP (quaternized poly(4-vinylpyridine) (QP4VP) and poly(4-cyanobiphenyl-4'-oxyundecylacrylate) (LCP)) was examined for protein and DNA detection. QP4VP-b-LCP was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Quaternization of P4VP with iodomethane (CH3I) made it a strong cationic polyelectrolyte and allowed QP4VP-b-LCP to form complexes with oppositely charged biological species. Several proteins, such as bovine serum albumin (BSA), hemoglobin (Hb), α chymotrypsinogen-A (ChTg), and lysozyme (LYZ), were tested for nonspecific protein detection. By injecting the protein solutions into the TEM grid cell, the initial homeotropic orientation of the TEM grid cell changed to a planar orientation above their isoelectric points (PIs) due to electrostatic interactions between QP4VP (+charge) and proteins (-charge), which did not occur below the PIs of the tested proteins. Their minimum concentrations at which the homeotropic to planar configurational change (H-P change) occurred were 0.01, 0.02, 0.03, and 0.04 wt.% for BSA, ChTg, Hb, and LYZ, respectively. One of the strong anionic polyelectrolytes, deoxyribonucleic acid (DNA) (due to the phosphate deoxyribose backbone) was also tested. A H-P change was observed with as little as 0.0013 wt.% salmon sperm DNA regardless of the pH of the cell. A H-P change occurred in 5CB and was observed by polarized optical microscopy. This simple and inexpensive setup for nonspecific biomaterial detection provides the basic idea for developing effective selective biosensors by introducing specific binding groups, such as the aptamer and antibody.

Topics: Animals; Biosensing Techniques; Biphenyl Compounds; Chymotrypsin; DNA; Electrolytes; Hemoglobins; Hydrocarbons, Iodinated; Isoelectric Point; Liquid Crystals; Magnetic Resonance Spectroscopy; Microscopy, Electron, Transmission; Muramidase; Nitriles; Optics and Photonics; Polymers; Polyvinyls; Salmon; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Static Electricity

In this study, we investigated the orientational behavior of liquid crystals (LCs) which is associated with the chitosan-disrupted phospholipid membrane at the aqueous/LC interface. The optical response of LCs changed from dark to bright after the transfer of an aqueous solution of chitosan onto the LC interface decorated with self-assembled monolayers of a negatively charged phospholipid, dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG). The chitosan-lipid interactions induced a rearrangement of the membrane, and thus, resulted in an orientational transition of LCs from a homeotropic to a planar state, thereby triggering a dark-to-bright shift in the optical response. We observed that LCs exhibited a bright-to-dark shift after an aqueous solution of lysozyme was transferred onto the chitosan-disrupted membrane, which implied that an enzymatic reaction between lysozyme and chitosan took place. We found that the addition of bovine serum album (BSA) induced a bright-to-dark change in the optical response; while LCs remained to appear bright after the transfer of chymotrypsin onto the aqueous/LC interface. We then further examined the interactions between other polyelectrolytes and phospholipid membranes.

Topics: Animals; Cattle; Chitosan; Chymotrypsin; Liquid Crystals; Membranes, Artificial; Microscopy, Polarization; Muramidase; Phosphatidylglycerols; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Static Electricity; Water

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

Many applications of nanobiomaterials rely on or are enhanced by specific, protein-mediated interactions with biological systems. These interactions can be engineered by chemically modifying the surface of the material to affect protein adsorption, or by altering the topography of the nanoscale surface. The covalent attachment or adsorption of proteins onto materials can greatly affect their structure and function, giving rise to either beneficial effects or to unpredictable and potentially undesirable effects. Thus, it is essential to develop a detailed understanding of how nanostructured surface characteristics, such as atomic-scale topography, surface energy, and chemical structure may affect protein adsorption, structure, function, and stability. Herein we observe that nanoparticle morphology and protein surface coverage affect the structure, activity, and stability of adsorbed lysozyme (Lyz) and α-chymotrypsin (ChT) in a manner that is protein specific. Wet chemical methods were used to synthesize gold nanocubes (AuNC) with {100} facets and gold nanooctahedra (AuNO) with {111} facets. Differences in adsorption on AuNC and AuNO are observed, which may be attributed to the atomic topography of the material. Nanoparticles, as well as the final form of the resulting protein conjugates, were thoroughly characterized through various physical, microscopic, and spectroscopic techniques. As a result, additional insight into the influence of nanoscale surface properties was obtained, which will enhance our fundamental understanding of how such properties affect protein structure and function, and will hence assist us in strategically engineering protein-nanomaterial conjugates for a variety of biomedical applications.

Topics: Adsorption; Chymotrypsin; Gold; Metal Nanoparticles; Muramidase; Nanotechnology; Protein Conformation; Proteins

Preferential interaction coefficients provide a thermodynamic measure to quantify the interactions between cosolutes and a protein. Preferential interactions of cosolutes can be measured experimentally using dialysis/densimetry and vapor pressure osmometry (VPO) techniques. The cosolute arginine is a widely used aggregation suppressor with a seemingly unique behavior. Its role in protein aggregation has been studied extensively, although a complete mechanistic understanding of its behavior is lacking. Moreover, due to experimental limitations, experimental preferential interaction data for arginine has only been reported at low concentrations. Schneider and Trout ( J. Phys. Chem. B 2009 , 113 , 7 ) have reported experimental preferential interaction data for argHCl (up to 0.7 m), and their study raised several interesting questions about the preferential interaction of arginine with proteins. Arginine is attracted to proteins at low concentrations but it was highly excluded at high concentrations. Furthermore, the preferential interaction coefficient values were found to vary as a square of the concentration, which is different from commonly observed linear relationship for other cosolutes like urea, glycerol, guanidinium hydrochloride, etc. In this study, preferential interaction coefficients of argHCl have been estimated computationally for two proteins (lysozyme and α-chymotripsinogen A) for a large concentration range (up to 2.8 m). On the basis of these results, the molecular level interactions responsible for the nonlinear exclusion of arginine from the protein surface are identified.

Topics: Arginine; Chymotrypsin; Muramidase; Protein Binding; Thermodynamics

Proteins (bovine serum albumin (BSA), α-chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L(-1) aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol-OT, or AOT, and a 1,3-dioxolane (or cyclic ketal) alkyl ethoxylate, CK-2,13-E5.6), producing a three-phase (Winsor-III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5-13 g L(-1)) and small in volume (12-20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W-III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK-2,13-E3 , or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α-chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α-chymotrypsin being >90% of its original value.

Topics: Chymotrypsin; Cytochromes c; Dioctyl Sulfosuccinic Acid; Emulsions; Models, Theoretical; Muramidase; Proteins; Serum Albumin, Bovine

Many biomedical applications of gold nanoparticles (NPs) rely on proteins that are covalently attached or adsorbed on the NP surface. The biological functionality of the protein-NP conjugate depends on the protein's ability to interact with target molecules, which is affected by NP characteristics such as size, curvature, aspect ratio, morphology, crystal structure, and surface chemistry. In the present study, the effect of gold nanoparticle morphology on the structure and function of adsorbed enzymes, lysozyme (Lyz) and α-chymotrypsin (ChT), has been investigated. Gold nanospheres (AuNS) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm. Under saturating conditions, proteins adsorb with a higher surface density on AuNR when compared to AuNS. In the case of Lyz, adsorption on AuNS and AuNR resulted in a 10% and 15% loss of secondary structure, respectively, leading to conjugate aggregation and greatly reduced enzymatic activity. ChT retained most of its secondary structure and activity on AuNS and AuNR at low surface coverages; however, as protein loading approached monolayer conditions on AuNR, a 40% loss in secondary structure and 86% loss of activity was observed. Subsequent adsorption of ChT in multilayers on the AuNR surface allowed the conjugates to recover activity and remain stable. It is clear that AuNP morphology does affect adsorbed protein structure; a better understanding of these differences will be essential to engineer fully functional nanobioconjugates.

Topics: Adsorption; Biocompatible Materials; Chymotrypsin; Gold; Materials Testing; Muramidase; Nanoparticles; Particle Size; Surface Properties

The high-throughput and sensitive characterization of native proteins in biological samples is of increasing interest in multiple disciplines. Extractive electrospray ionization (EESI) forms ions of native proteins including lysozyme, α-chymotrypsin, myoglobin, human serum albumin, RNAse A and blood hemoglobin in extremely complex biosamples or PBS buffer solutions by softly depositing charges on the protein molecules. This method produces no significant conformational changes of the proteins in the ion formation process, and features direct detection of trace proteins present in biological matrices. The detection limit of low pmol L(-1) for lysozyme in untreated biological liquids such as human urine and tears was demonstrated using EESI mass spectrometry (MS), showing an attractive MS platform for the direct analysis of native proteins in actual biological samples.

Topics: Body Fluids; Chymotrypsin; Hemoglobins; Humans; Ions; Muramidase; Myoglobin; Proteins; Ribonucleases; Serum Albumin; Spectrometry, Mass, Electrospray Ionization

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.

Topics: Adsorption; Chymotrypsin; Humans; Immobilized Proteins; Immunoglobulin G; Lasers; Microscopy, Atomic Force; Muramidase; Myoglobin; Nanostructures; Protein Array Analysis; Serum Albumin; Surface Properties; Temperature

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.

Topics: Adsorption; Benzylamines; Binding Sites; Chelating Agents; Chromatography, Affinity; Chymotrypsin; Cytochromes c; Hydrogen-Ion Concentration; Ligands; Molecular Structure; Muramidase; Ribonuclease, Pancreatic; Sensitivity and Specificity; Silicon Dioxide; Surface Properties; Tetrazoles; Zinc

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

Topics: Animals; Carbodiimides; Chymotrypsin; Cytochromes c; Drug Carriers; Drug Delivery Systems; Esterification; Hydrogen-Ion Concentration; Hydrolysis; Lactoglobulins; Lipase; Malates; Methylation; Microscopy, Electron, Scanning; Muramidase; Myoglobin; Nanospheres; Particle Size; Physarum polycephalum; Polymers; Proteins; Serum Albumin, Bovine; Static Electricity; Surface Properties

Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, alpha-chymotrypsin, peroxidase and beta-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.

Topics: Animals; beta-Galactosidase; Chemical Precipitation; Chemistry, Pharmaceutical; Chymotrypsin; Drug Carriers; Drug Compounding; Enzyme Stability; Enzymes; Kinetics; Lactic Acid; Microspheres; Muramidase; Oils; Peroxidase; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protein Conformation; Sodium Chloride; Solubility; Solvents; Technology, Pharmaceutical; Water

To investigate globular protein-protein and protein-salt interactions in electrolyte solutions, a potential of mean force including hard-core repulsion, van der Waals attraction and electric double layer repulsion is proposed in this work. Both van der Waals attraction and double-layer repulsion are represented using hard spheres with two-Yukawa tails. The explicit analytical solution of osmotic pressure is derived from the first-order mean spherical approximation. From the comparison between the calculated and experimental values of osmotic pressures for aqueous bovine serum albumin (BSA), lysozyme, alpha-chymotrypsin, and immuno-gamma-globulins (IgG) solutions, we found that the proposed model is adequate for the description of the interactions between proteins at low ionic strength and small self-association of protein molecules. At high ionic strength, the charge inversions of protein molecules should be taken into account.

Topics: Binding Sites; Chymotrypsin; Electrolytes; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Models, Biological; Models, Molecular; Muramidase; Osmolar Concentration; Osmotic Pressure; Protein Binding; Salts; Serum Albumin, Bovine; Solutions; Surface Properties; Thermodynamics; Water

Topics: Animals; Chickens; Chymotrypsin; Computers; Crystallography; Eggs; Muramidase; Photography

The equation of state (EOS) of Duh and Mier-y-Terán for one Yukawa potential is expanded to two Yukawa potentials to describe the nonidealities of the charged lysozyme and alpha-chymotrypsin solutions. Instead of the classical DLVO theory, the attractive dispersion and double-layer repulsion interactions are represented by two Yukawa potentials, respectively. For the aqueous lysozyme solutions, the only adjustable dispersion energy parameter epsilon/k is regressed and the average deviation is 1.76%. For the aqueous alpha-chymotrypsin solutions, two adjustable parameters (the molecular weight and dispersion energy parameter) are regressed and the average deviation is 7.62%. Some correlation and prediction results are discussed.

Topics: Chymotrypsin; Electrolytes; Kinetics; Muramidase; Osmotic Pressure

Beads prepared from a thermosensitive polymer, hydroxypropylcellulose, exhibit temperature-dependent porosity. At temperatures below 40 degrees C the beads are swollen having large pores, while at temperatures above 45 degrees C the beads are in a shrunken state having smaller pores. In the presence of 1 M NaCl the transition temperature decreased to about 30 degrees C. In a swollen state the size of pore is large enough to accommodate lysozyme (mol. mass 14400) and alpha-chymotrypsin (mol. mass 21600) but not bovine serum albumin (mol. mass 67000). When the beads are shrunken, all the proteins are eluted from the column packed with hydroxypropylcellulose beads in the volume close to the void volume of the column.

Topics: Chromatography, Gel; Chymotrypsin; Hot Temperature; Muramidase; Serum Albumin, Bovine; Temperature

Topics: Amino Acids; Cesium Isotopes; Chromatography; Chymotrypsin; Deuterium; Muramidase; Proteins; Radiation Effects; Research; Serine; Tryptophan