Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Capillary electrophoresis (CE) was investigated for characterizing poly(ethylene glycol) (PEG) attachment ("PEGylation") and PEG removal ("dePEGylation") of proteins. Lysozyme was used as a model protein because it is one of the best understood enzymes, has a high ionic strength (high pI value; thus making it suitable for CE), and has a tertiary structure that is known with high resolution. Several PEG derivatives, both hydrolytically degradable and nondegradable and with varying reactivities toward amino groups, were used to couple to amino groups (six epsilon-amino and one alpha-amino) on the surface of the protein. Capillary electrophoresis was found to be useful in following both the PEGylation and dePEGylation of lysozyme. Capillary electrophoresis separation is based on the size of the conjugate, which is determined by the number and molecular weight of the PEG that is attached. Baseline resolution was obtained between the peaks for each PEG chain attached per protein molecule ("PEGmers") for PEG molecular weights >5000, although individual PEGmers could be recognized at lower molecular weights without baseline separation. Highly modified lysozyme showed complete inactivation, but when released from the degradable PEG, regained >60% of the original in vitro activity. The sites of PEGylation were determined using a tryptic map of the modified and unmodified protein. Typically, peptide fragments are separated by reversed-phase HPLC, but we show that CE can provide a complementary separation technique for determining sites of PEGylation. Capillary electrophoresis has advantages of high efficiency separations, rapid analysis, and ease of use.
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PMID:Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy. 981 3

Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities. For drugs bearing one or more amino groups, reductive amination is a potentially useful method for conjugation to PEG. PEG acetaldehyde has been used for this purpose, but its ease of polymerization under certain conditions and its susceptibility to air oxidation have caused some problems in its application. A simple and reliable method for preparation and use in reductive amination of PEG acetaldehyde hydrate generated in situ by hydrolysis of PEG acetaldehyde diethylacetal is demonstrated. PEG acetaldehyde diethylacetal is prepared in high yield and purity by reaction of PEG with chlorodiethylacetal in dioxane in the presence of finely powdered sodium hydroxide under heterogeneous conditions. PEG acetaldehyde hydrate is generated in solution by hydrolysis in aqueous acids. Solutions of the hydrate may be used directly, in conjunction with sodium cyanoborohydride, to effect reductive amination. We demonstrate application of these methods in PEGylation of lysozyme and chitosan to form water-soluble methoxy poly(ethylene glycol) (mPEG) derivatives and PEG-chitosan hydrogels.
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PMID:Reductive amination using poly(ethylene glycol) acetaldehyde hydrate generated in situ: applications to chitosan and lysozyme. 981 4

A new method of formation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumin) under high pressure has been developed. The existence of polymer in complexes was proved using 3H-labeled PEG. Complexes between PEG and ChT were studied in detail. It was shown that the composition of complexes (the number of polymer chains per ChT molecule) depends on the molecular mass of PEG and decreases with the increase of molecular mass from 300 to 4000. At the same time, the portion of the protein (wt. %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt. %. It was shown that kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical to the corresponding values for the native ChT. The conformational properties of ChT in complexes were studied by circular dichroism. It was shown that the enzyme in complexes fully retains its secondary structure. The estimation of steric availability of PEG polymer chains in complexes was evaluated by the complexation with alpha-cyclodextrin (CyD). It was shown that in contrast to free PEG, only part (approximately 10%) of PEG polymer chains in PEG--ChT complexes participate in the complexation with CyD. Hence, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule in a region far from the active site of the enzyme and results in the significant decrease in the mobility of polymer chains.
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PMID:Noncovalent complexes between poly(ethylene glycol) and proteins. 986 73

A universal procedure to isolate cross-linked peptides has been demonstrated. The procedure relies on initially converting the epsilon-amino groups of lysine to dimethyl lysine by reductive methylation with sodium cyanoborohydride and formaldehyde. The lysine-modified protein is proteolytically cleaved and the resulting alpha-amino groups derivatized with methoxypoly(ethylene glycol)5000 succinyl hydroxysuccinimide. Any unintentional derivatization of tyrosine side chains can be reversed by incubation under mildly alkaline conditions. The cross-linked polypeptides contain two poly(ethylene glycol)5000 chains while non-cross-linked peptides contain a single poly(ethylene glycol)5000 chain. The two populations of peptides can be separated by gel filtration chromatography. This procedure has been shown capable of isolating cross-linked peptides using glutathione, lysozyme, chemically cross-linked hemoglobin, and neurofibrillary tangles isolated from the brain of a case of Alzheimer's disease.
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PMID:Universal isolation of cross-linked peptides: application to neurofibrillary tangles. 989 72

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiolates on gold generated from the tri(ethylene glycol)-terminated thiol 1 (HS(CH2)11(OCH2CH2)3OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-terminated thiol2(HS(CH2)11(OCH2-CH2)6OCH2CO2H) (chi(2) = 0.0-1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to nonspecific adsorption of proteins having a wide range of molecular weights and isoelectric points. The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized. For the proteins investigated in detail--carbonic anhydrase and lysozyme--the highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pI) of the protein, at a concentration of approximately 0.5 mg mL-1. Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of Kd, koff, and kon.
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PMID:A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: a surface plasmon resonance study. 1005 46

Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components. We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments.
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PMID:Contribution of protein charge to partitioning in aqueous two-phase systems. 1009 60

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.)
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PMID:Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies. 1009 1

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.
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PMID:Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components. 1035 68

A novel polypeptide hydrogel has been synthesized by crosslinking poly(L-glutamic acid) (PLG) with poly(ethylene glycol) (PEG). The PLG-PEG hydrogel was shown to be highly hydrophilic, and the extent of swelling varied with pH, increasing at higher ionization of the PLG. Aside from electrostatic effects, such as ion-ion repulsion and internal ion osmotic pressure, circular dichroism studies showed that swelling response to pH also is affected by secondary structural attributes associated with the polypeptide backbone. Modification of the polypeptide by changing its hydrophobicity and degree of ionization was an effective method for altering the overall extent of pH-responsive swelling. Rapid de-swelling (contraction) was observed when the PLG-PEG hydrogel was transferred from high to low pH buffer solution, and this swelling/de-swelling behavior was reversible over repeated cycles. Drug release from swollen hydrogels was examined using the model protein lysozyme. Rapid de-swelling of the hydrogel was found to be an effective means of facilitating lysozyme release. The crosslinking of synthetic polypeptides with PEG appears to be a highly versatile approach to the preparation of pH-responsive biodegradable hydrogels.
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PMID:A pH- and ionic strength-responsive polypeptide hydrogel: synthesis, characterization, and preliminary protein release studies. 1049 96

The properties of a series of multiblock copolymers, based on hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(butylene terephthalate) (PBT) blocks were investigated with respect to their application as a matrix for controlled release of proteins. The degree of swelling, Q, of the copolymers increased with increasing PEG content and with increasing molecular weight of the PEG segment. Within the composition range tested, Q varied from 1.26 for polymers with PEG segments of 600 g/mol and a PBT content of 60 weight.% up to 3.64 for polymers with PEG segments of 4000 g/mol and a PEG/PBT weight ratio of 80:20. Equilibrium stress (compression)-strain measurements were performed in order to estimate mesh sizes. The mesh size of the copolymers ranged from 38 to 93 A, which was experimentally confirmed by diffusion of vitamin B(12) (hydrodynamic diameter d(h)=16.6 A), lysozyme (d(h)=41 A) and bovine serum albumin (d(h)=72 A). The in vitro degradation of PEG/PBT copolymers with a PEG block length of 1000 g/mol and PEG/PBT weight ratios of 70:30, 60:40 and 40:60 was studied. Matrices with increasing PEG contents exhibited a faster weight loss in phosphate-buffered saline (pH 7.4) at 37 degrees C. Over a degradation period of 54 days, M(n) decreased by about 35-45%, while the composition of the matrices, determined by NMR, remained almost constant.
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PMID:A controlled release system for proteins based on poly(ether ester) block-copolymers: polymer network characterization. 1052 76


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