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)

Immobilized metal affinity chromatography (IMAC) was investigated for its ability to characterize the histidine-related surface structure of a protein, that is, a histidine residue's surface accessibility and its potential involvement in intramolecular interactions. T4 lysozyme was chosen as the model protein. Seven amino acid sites were selected on the basis of their relative surface accessibility, and they were substituted with histidine via site-directed protein mutagenesis to generate seven T4 lysozyme variants, each containing only one histidine residue on its surface, with various surface accessibility. IMAC was then used to experimentally quantify the interaction of each lysozyme variant with immobilized copper ions. A direct correlation was shown between the protein binding affinity and the surface accessibility of the histidine residue. Of all the lysozyme variants, K83H and K147H showed unusually low binding strength, as compared with variants having a histidine residue with a similar surface accessibility. However, with the aid of molecular modeling, their relatively low binding affinities were predicted to be the result of the involvement of the histidine residue in intramolecular interactions. In contrast to previously reported results, our results showed that lysozyme still binds to the IMAC column, even if its histidine residue is involved in intramolecular bonding, such as a hydrogen bond, albeit at reduced strength, as compared with the variant containing a histidine residue with a similar surface accessibility.
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PMID:Correlation between protein binding strength on immobilized metal affinity chromatography and the histidine-related protein surface structure. 1680 52

In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO(2) for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23.
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PMID:Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method. 1689 11

Trivalent and pentavalent arsenic were incubated with sulfur-containing amino acid, peptide and protein solutions both as organic compounds (phenylarsine oxide, phenylarsonic acid, dimethylarsinic acid, monomethylarsonic acid) and as inorganic compounds (arsenite, As(III), and arsenate, As(V)). After incubation of phenylarsine oxide solutions with cysteine and glutathione the mass spectra showed a covalent bond between arsenic and sulfur, which was stable at both acidic and neutral pH values. The mass spectra were dominated by monovalent ions at m/z 272 for cysteine samples and at m/z 458 for glutathione samples. Based on these masses the ionic structures could be ascribed to either fragment ions of the covalent arsenic-sulfur complexes or to other arsenic-bonding sites presumably at the amino group. Interestingly, under the same conditions no interactions of inorganic arsenite or arsenate could be measured. In the presence of added Cu(2+) ions all mass signals caused by a reaction of phenylarsine oxide with glutathione disappeared. In these mass spectra only the oxidised form of glutathione (GSSG) was found because of the redox activity of Cu(II). For the model protein lysozyme, no interactions with arsenic could be detected, whereas definite Cu- and Zn-lysozyme complexes with a stoichiometry of 1:1 and 2:1 for Zn(2+) ions and Cu(2+) ions, respectively, were observed. In contrast, for thioredoxin a bonding of As that depended on the concentration of the disulfide-reducing agent tris(2-carboxyethyl) phosphine was demonstrated. For three different phenylarsonic acids and for dimethylarsinic acid that all contain pentavalent arsenic, complexes with glutathione appeared in the mass spectra, which can be attributed to non-covalent interactions or to a covalent bond caused by an additive reaction. The optimisation of the experimental conditions necessary for the mass spectrometric analysis of the interactions of the arsenic species with peptides and proteins is described and the obtained mass spectra that provide information on the kinds of bonds are discussed.
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PMID:Mass spectrometric evidence for different complexes of peptides and proteins with arsenic(III), arsenic(V), copper(II), and zinc(II) species. 1715 58

A novel light-sensitive and cation-exchange copolymer (PNBCC) has been synthesized by random copolymerization of chlorophyllin sodium copper salt, crylic acid, n-butyl acrylate, and N-isopropylacrylamide. The PNBCC copolymer showed reversible dissolution and could be precipitated by 488 nm laser irradiation with the least light density of 1.70 x 10(5) W/m(2). By optimizing the ratio of monomers, pH, and ion concentration, over 95% copolymer was recovered by laser irradiation. The copolymer was used to purify lysozyme as light-sensitive cation exchanger, and its adsorption matched a Langmuir adsorption isotherm with maximum adsorption capacity of 98,900 U/g and dissociated constant of 852 U/mL. By applying the copolymer to the separation of lysozyme from egg white, the specific activity of lysozyme was improved from 399 to 6346 U/mg and the recovery of lysozyme achieved 81.3%.
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PMID:Preparation of a light-sensitive and reversible dissolution copolymer and its application in lysozyme purification. 1788 Jan 2

The mixed-ligand copper(II) complex [Cu(tdp)(tmp)](ClO4), where H(tdp) is 2-[(2-(2-hydroxyethylamino)ethylimino)methyl]phenol and tmp is 3,4,7,8-tetramethyl-1,10-phenanthroline, exhibits cleavage of the proteins bovine serum albumin and lysozyme, producing approximately 5 and 4 kDa protein fragments respectively within a few minutes at micromolar concentrations. The hydrophobic tmp ligand recognizes the hydrophobic site and enhances protein binding and cleavage even at physiological pH and temperature.
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PMID:Cleavage of proteins by a mixed-ligand copper(II) phenolate complex: hydrophobicity of the diimine co-ligand promotes cleavage. 1799 46

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80-100 degrees C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.
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PMID:Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates. 1815 58

High-resolution solid-state NMR (SSNMR) of paramagnetic systems has been largely unexplored because of various technical difficulties due to large hyperfine shifts, which have limited the success of previous studies through depressed sensitivity/resolution and lack of suitable assignment methods. Our group recently introduced an approach using "very fast" magic angle spinning (VFMAS) for SSNMR of paramagnetic systems, which opened an avenue toward routine analyses of small paramagnetic systems by (13)C and (1)H SSNMR [Y. Ishii et al., J. Am. Chem. Soc. 125, 3438 (2003); N. P. Wickramasinghe et al., ibid. 127, 5796 (2005)]. In this review, we discuss our recent progress in establishing this approach, which offers solutions to a series of problems associated with large hyperfine shifts. First, we demonstrate that MAS at a spinning speed of 20 kHz or higher greatly improves sensitivity and resolution in both (1)H and (13)C SSNMR for paramagnetic systems such as Cu(II)(DL-alanine)(2)H(2)O (Cu(DL-Ala)(2)) and Mn(acac)(3), for which the spectral dispersions due to (1)H hyperfine shifts reach 200 and 700 ppm, respectively. Then, we introduce polarization transfer methods from (1)H spins to (13)C spins with high-power cross polarization and dipolar insensitive nuclei enhanced by polarization transfer (INEPT) in order to attain further sensitivity enhancement and to correlate (1)H and (13)C spins in two-dimensional (2D) SSNMR for the paramagnetic systems. Comparison of (13)C VFMAS SSNMR spectra with (13)C solution NMR spectra revealed superior sensitivity in SSNMR for Cu(DL-Ala)(2), Cu(Gly)(2), and V(acac)(3). We discuss signal assignment methods using one-dimensional (1D) (13)C SSNMR (13)C-(1)H rotational echo double resonance (REDOR) and dipolar INEPT methods and 2D (13)C(1)H correlation SSNMR under VFMAS, which yield reliable assignments of (1)H and (13)C resonances for Cu(Ala-Thr). Based on the excellent sensitivity/resolution and signal assignments attained in the VFMAS approach, we discuss methods of elucidating multiple distance constraints in unlabeled paramagnetic systems by combing simple measurements of (13)C T(1) values and anisotropic hyperfine shifts. Comparison of experimental (13)C hyperfine shifts and ab initio calculated shifts for alpha- and beta-forms of Cu(8-quinolinol)(2) demonstrates that (13)C hyperfine shifts are parameters exceptionally sensitive to small structural difference between the two polymorphs. Finally, we discuss sensitivity enhancement with paramagnetic ion doping in (13)C SSNMR of nonparamagnetic proteins in microcrystals. Fast recycling with exceptionally short recycle delays matched to short (1)H T(1) of approximately 60 ms in the presence of Cu(II) doping accelerated 1D (13)C SSNMR for ubiquitin and lysozyme by a factor of 7.3-8.4 under fast MAS at a spinning speed of 40 kHz. It is likely that the VFMAS approach and use of paramagnetic interactions are applicable to a variety of paramagnetic systems and nonparamagnetic biomolecules.
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PMID:Progress in 13C and 1H solid-state nuclear magnetic resonance for paramagnetic systems under very fast magic angle spinning. 1826 15

Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(L)-histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA-MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA-MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.
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PMID:Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels. 1839 14

Three new IMAC chelating systems, incorporating immobilised xylenyl-bridged bis(1,4,7-triaza-cyclonane) ligands, complexed with Cu(2+) ions to form binuclear species, have been prepared. Their binding properties have been investigated with three small globular proteins (hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome c). The effects of buffer pH, ionic strength and composition on the binding behaviour of these proteins to these new IMAC sorbents have been examined and compared with those found for the corresponding immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). Higher protein binding affinities were observed with the Cu(2+)-bis(tacn) sorbents compared to the Cu(2+)-tacn system, consistent with the immobilized binuclear copper(II) species undergoing enhanced coordinative interaction with the surface-exposed histidine residues of these proteins. Moreover, the protein binding characteristics of these IMAC sorbents at higher ionic strengths, such as 1M NaCl, also reflect the presence of the aromatic ring in the bis(tacn) ligands, whereby hydrophobic pi/pi stacking interactions can occur with the proteins.
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PMID:Examination of the binding behaviour of several proteins with the immobilized copper(II) complexes of o-, m- and p-xylylene bridged bis(1,4,7-triazacyclononane) macrocycles. 1844 7

Relaxometric studies are still of scientific interest due to their use in medicine and biology. In this study, proton T(1) and T(2) relaxivities of Mn(II), Cu(II) and Cr(III) in water were determined in the presence and absence of various proteins (albumin, alpha-globulin, gamma-globulin, lysozyme, fibrinogen). The 1/T(1) and 1/T(2) in all solutions are linearly proportional to the concentration of the paramagnetic ions. Mn(II) has the great influence to alter relaxations in all protein solutions, while Cu(II) and Cr(III) have a poor influence on the relaxations. In addition, Mn(II) and Cu(II) are bound to each protein, but Cr(III) is not bound to any protein.
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PMID:Determination of proton relaxivities of Mn(II), Cu(II) and Cr(III) added to solutions of serum proteins. 1938 83


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