Absolute and relative quantification of sheep brain prion protein (PrP) allelic variants by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation in the tissues of affected individuals of an abnormal form (PrP(Sc)) of a protein naturally produced by the host, the cellular prion protein (PrP(C)). In sheep, susceptibility to TSEs is tightly controlled by polymorphism at positions 136 (A or V), 154 (R or H) and 171 (R or Q) of the Prnp gene encoding the prion protein (PrP). Quantification of PrP variants at positions 136, 154 and 171 can be achieved by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of the respective peptides 114-139, 152-159 and 160-171 obtained after tryptic digestion of the PrP protein. In this study we quantified the tryptic peptide 114-139 containing the first polymorphic site. Quantification was either relative, between variants of this peptide, or absolute with respect to the C-terminally (18)O-labelled peptide obtained by hydrolysing known amounts of recombinant protein with trypsin in H(2) (18)O. After purification of PrP(C) and PrP(Sc) from the brain of two heterozygous sheep carrying either the ARQ/VRQ or ARR/VRQ genotypes, the proportion of each variant was measured. In the ARQ/VRQ animal, while both variants were equally represented in the normal isoform, the VRQ variant was predominantly found in the abnormal PrP protein, suggesting dissimilar behaviour of the two variants in the pathological process. The situation was even more contrasted in the ARR/VRQ animal where PrP(Sc) was solely composed of the VRQ variant. These two examples clearly illustrate the value of MALDI-TOF analysis, combined with appropriate immunopurification techniques, in seeking a precise understanding of the influence of PrP polymorphisms on TSE pathogenesis.     

Detection of single-nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis

An alternative method is described for the determination of ovine prion protein allelic variants at codon 136, 154, and 171. The four mutations responsible for amino acid changes are typed simultaneously. The technique utilizes dideoxy chain termination reaction using fluorescently labeled dideoxy nucleotides. The single-base extended primers are resolved on a capillary electrophoresis instrument. Data obtained by our approach are presented according to genotype distribution in some breeds as a part of the validation procedure.     

Analysis of local polarity change around Cys34 in bovine serum albumin during N→B transition by a polarity-sensitive fluorescence probe

The change trend of the local environment of Cys34 domain in bovine serum albumin has been studied as a function of pH value by using thiol-specific and polarity-sensitive fluorescent probe 3-(4-chloro-6-p-maleimidylphenoxyl-1,3,5-triazinylamino)-7-dimethylamino-2-methyl-phenazine. The local polarity of the Cys34 domain is found to rise with the increase of pH values, and the corresponding dielectric constant is raised from 12.8 at pH 6.0 to 23.3 at pH 9.1. The result shows that the environment of the Cys34 domain is rather hydrophobic in normal state at pH 6.0 and becomes a little hydrophilic in the course of N→B transition, which may be attributed to the slight unfolding of the protein and thus the increasing of exposure of the previously relatively buried Cys34. In addition, the increased dielectric constant (23.3) is much lower than that (80.1) of water, suggesting that the unfolding of bovine serum albumin does not cause the full exposure of the Cys34 to the aqueous media during the transition.     

Hierarchical Unfolding of Mj HSP16.5

Mj HSP16.5 is a small heat shock protein (sHSP) from the hyperthermophilic methanoarchaeon, Methanococcus jannaschii (Mj), which lives at the environment of high temperature up to 94 °C. The structural data showed that Mj HSP16.5 was a 24-mer that formed a hollow sphere with octahedral symmetry. Mj HSP16.5 was very stable at pH 7 that it maintained the 24-mer structure even at 85 °C. In the present study, we investigated the unfolding process of Mj HSP16.5 in the presence of denaturants using several techniques, including circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and size exclusive chromatography (SEC). We found that 8 mol·L⁻¹ urea had no obvious effect on the structure of Mj HSP16.5 at pH 7. The unfolding of Mj HSP16.5 at pH 7 in the presence of guanidine hydrochloride (GdHCl) showed hierarchical behavior. Three significant transitions were observed around 2.0, 3.0, and 6.0 mol·L⁻¹ GdHCl at pH 7. ANS (8-anilino-1- naphthalenesulfonic acid) titration results showed that the binding ability of Mj HSP16.5 to ANS decreased gradually as the concentration of GdHCl increased until around 2.0 mol·L⁻¹ GdHCl, indicating surface hydrophobic area change, and this first transition was companioned with precipitation of Mj HSP16.5. Acrylamide quenching of fluorescence showed that the Stern-Volmer constant changed at about 3.0 mol·L⁻¹ GdHCl, indicating changes of the dimeric interface, and this phase transition was companioned with oligomeric state change from 24-mer to small oligomers (4-mer to 8-mer). The last unfolding phase started around 5.0 mol·L⁻¹ GdHCl, with a midpoint of 6.1 mol·L⁻¹ GdHCl, and Mj HSP16.5 was completely unfolded at 7.0 mol·L⁻¹ GdHCl. We also found that Mj HSP16.5 could be quite easily unfolded at pH 3, where it could be completely unfolded in 4.0 mol·L⁻¹ GdHCl.     

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.     

Preparation of gold nanoparticles on eggshell membrane and their biosensing application

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25 ± 7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl₄ without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl₄) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH ≤3.0 and ≥7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO_x) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO_x. The linear response range of the glucose biosensor is 20 μM to 0.80 mM glucose with a detection limit of 17 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.     

Site-selective probe for investigating the asynchronous unfolding of domains in bovine serum albumin

A convenient method is proposed for precise investigation of the asynchronous structural transition of the domains in bovine serum albumin (BSA) during unfolding process. The method is based on a site-selective probe, alizarin red S (ARS), which has a high affinity to the subdomain IIA of BSA. BSA-ARS complex was formed and gradually unfolded by urea from 0 to 8.0 M. The unfolding occurred in different domains of BSA resulted in distinct alterations of the microenvironment of the bound ARS. The spectral response of BSA-ARS complex, including the color, the UV absorption at 530 and 432 nm, and the intrinsic fluorescence at 342 and 310 nm with the excitation wavelength of 280 nm, showed slight changes in the urea concentration from 0 to 4.5 M, drastic changes from 4.5 to 6.0 M, and almost no changes from 6.0 to 8.0 M. The redox behavior of bound ARS between 0.3 and 0.8 V also showed the same trend. Consequently, a two-step, three-state transition process was monitored by naked eyes, UV-vis spectroscopy and electrochemistry. It is the first report to realize the indicator of the intermediate state during the unfolding process of BSA through convenient methods instead of expensive approaches. The work provides a facile method for the investigation of the unfolding process of multidomain proteins.     

Studies on the photophysical characteristics of poly(carboxylic acid)s bound protoporphyrin IX and metal complexes of protoporphyrin IX

The copolymers of methacrylic acid with protoporphyrin IX (PPIX) and the metal complexes, zinc protoporphyrin IX and magnesium protoporphyrin IX were synthesised and characterised. Corresponding acrylic acid copolymers were also synthesised. The steady state absorption and fluorescence spectral properties of the macromolecular bound fluorophores PPIX, Zn-PPIX and Mg-PPIX were investigated. Poly(methacrylic acid) bound protoporphyrin IX, zinc protoporphyrin IX and magnesium protoporphyrin IX show an increase in the fluorescence intensity and lifetime with increase in the pH in the range 2-8 with a marked transition around pH 6.0-7.0. The fluorophore concentration in the dilute solution of the copolymers is micromolar and the fluorophore to the carboxylic acid monomer ratios in the copolymer is around 10⁻³. The molecular weight of the copolymers is 100 ± 10 kD. The fluorescence decay curves of all the fluorophore bound polymers follow biexponential decay fit independent of pH. Poly(MAA-co-PPIX) and poly(MAA-co-MgPPIX) undergo well marked pH induced structural transitions in the pH range of 6.0-7.0 whereas poly(MAA-co-ZnPPIX) undergoes pH induced structural transitions in the pH range of 4.0. In the case of polyacrylic acid copolymers the changes observed in the steady state and time resolved fluorescence studies are less marked. The distinct hydrophobic and hydrophilic environments experienced by the fluorophore bound to PMMA are attributed to the dynamics of the macromolecules in dilute aqueous solutions manifested by the α-methyl group present in the copolymer. The studies carried out using the fluorophores in the time windows from 2 ns to 12 ns indicate evolving trends in the dynamic coiling and reverse coiling of poly methacrylic acid chain.     

Production of carrier-free radioisotopes from targets irradiated with deuterons or alpha particles in the U-120 Cracow Cyclotron

The results of experiments on the preparation of carrier-free lanthanides from some targets irradiated in the U-120 cyclotron are presented. In the reactions¹⁵¹Eu(α, n)^154m,154Tb,¹⁵¹Eu(α, 2n)¹⁵³Tb,¹⁵⁵Gd(α, 2n)¹⁵⁷Dy,¹⁶⁹Tm(α, n)¹⁷²Lu and¹⁶⁹Tm(α, 2n)¹⁷¹Lu, the isotopes^154m,154+153Tb,¹⁵⁷Dy and^172+171Lu were obtained. These were seaprated by means of ion-exchange chromatography. Part II: see Ref. 2     

小分子热休克蛋白Mj HSP16.5的分级变性

应用荧光光谱、圆二色光谱、体积排阻色谱、激光动态光散射等技术, 研究了来自嗜热古细菌Methanococcus jannaschii (Mj)的小分子热休克蛋白Mj HSP16.5在变性剂作用下的变性过程. 研究表明, 在pH 7时, Mj HSP16.5在8 mol·L-1尿素作用下不会发生变性. 在pH 7条件下, 盐酸胍对Mj HSP16.5的变性表现为一个分级过程,分别在2.0、3.0和6.0 mol·L-1盐酸胍浓度附近,出现明显的结构变化; 到7.0 mol·L-1盐酸胍时, Mj HSP16.5才完全变性. 降低溶液pH值将使Mj HSP16.5的变性变得更为容易.     

4-Chlorophenol degradation by chloroperoxidase from Caldariomyces fumago

This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl⁻ containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5–50 and 0.005–50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H₂O₂ (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.     

Complexometric Titration of Metals Using a Lead Selective Electrode

Abstract

A solid state lead selective electrode is used satisfactorily to indicate the end point eufing the complexometric titration of Ni, Co, Zn and V (IV) with EDTA, CDTA, and DTPA, in presence of a small amount of the corresponding lead complex. The optimum pH for the titration was in the range of 4.0 to 6.0. The quantity of lead complex used was variable and should be determined in each case. The best results are obtained when the metals being determined are present in the range of 25 to 50 micromoles. Nickel, cobalt, and zinc give good results with DTPA at pH 6.0; vanadium(IV) gives excellent results with any of the three reagents in the pH range of 4.0 to 6.0.     

Eco1524I, a type II restriction endonuclease

Various strains of Escherichia coli, isolated from different patients, were screened for type II restriction endonuclease activity. In 1 out of 23 patients, a type II restriction endonuclease activity was found. The restriction endonuclease designated Eco1524I was purified to near homogeneity, based on hydroxyapatite and heparin sepharose chromatography. Eco1524I exhibited endonuclease restriction activity in the pH range from 6.0 to 10.0 (maximum level at pH 8.0) and required Mg²⁺ as divalent cation. The enzyme was stable till temperature 55°C and pH range from 6.0 to 10.0. Eco1524I recognized the sequence 6-bp palindromic 5′AGG↑CCT 3′, producing blunt end and is found to be an isoschizomer of Stu I.     

Capillary electrophoresis of the scrapie prion protein from sheep brain

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrP^sc) that has a molecular mass of ca. 27 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrP^sc. The overall concentration of PrP^sc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrP^sc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrP^sc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrP^sc. After treatment with proteinase K and non-ionic detergents, PrP^sc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep. Capillary electrophoresis can be used to show immunocomplex formation when PrP^sc is present in sheep brain.     

Studies on Productivity and Characterization of Polygalacturonase from <Emphasis Type="Italic">Aspergillus giganteus</Emphasis> Submerged Culture Using Citrus Pectin and Orange Waste

Polygalacturonases are part of the group of enzymes involved in pectin degradation. The aim of this work was to investigate some of the factors affecting polygalacturonase production by an Aspergillus giganteus strain and to characterize this pectinolytic activity. Several carbon sources, both pure substances and natural substrates, were tested in standing cultures, and the best results were obtained with orange bagasse and purified citrus pectin. On citrus pectin as sole carbon source, the highest extracellular activity (9.5 U/ml and 40.6 U/mg protein) was obtained in 4.5-day-old cultures shaken at 120 rpm, pH 3.5 and 30°C, while on orange bagasse, the highest extracellular activity (48.5 U/ml and 78.3 U/mg protein) was obtained in 3.5-day-old cultures shaken at 120 rpm, pH 6.0 and 30°C. Optimal polygalacturonase activity was observed in assays conducted at pH 5.5–6.5 and 55–60°C. The activity showed good thermal stability, with half-lives of 90 and 30 min when incubated at 55 and 60°C, respectively. High stability was observed from pH 4.5 to 8.5; more than 90% of the activity remained after 24 h in this pH range.     

The Kinetics and mechanisms of oxidation of dihydroxybenzenes by ethylenediaminetetraacetatomanganate(III)

Summary The kinetics and mechanism of oxidation of 1,4-dihydroxybenzene (H₂Q), 2,5-dihydroxybenzenesulphonic acid (H₂SQ) and 4,5-dihydroxy-1,3-benzenedisulphonic acid (H₂T) to the correspondingortho orPara-quinones by ethylenediaminetetraacetatomanganate (III) has been investigated over the 2.5–6.0 pH range, at 20.0° by the stopped-flow technique. Complex behaviour is observed for H₂Q due to the superposition of the precursor complex formation and the redox act, while at higher pH's these reactions separate. For the other substrates, a single second order step (first order in each reactant) was present whose rate showed different pH dependences. All these features are discussed with reference to the reduction potential of the substrates, their acidity and coordinating ability.     

Investigation of trace level binding of PtCl6 and PtCl4 to alfalfa biomass (Medicago sativa) using Zeeman graphite furnace atomic absorption spectrometry

Batch laboratory experiments were performed to investigate the effects of pH, chemical modification, time dependency, and interference studies on the binding of trace concentrations of hexachloroplatinate(IV) and tetrachloroplatinate(II) to alfalfa biomass. The pH profiles were measured between pH 2.0 and 6.0. It was found that the binding of trace concentrations of platinum(IV and II) to alfalfa biomass was dependent on pH with a maximum binding occurring at pH 3.0 and a minimum at pH 6.0. When the alfalfa biomass was chemically modified (esterified), maximum binding occurred at pH 6.0 for both oxidation states of platinum. From the batch time dependency experiments, it was found that binding took at least 20 min to level off for both platinum oxidation states. Batch experiments were performed with various concentrations of calcium, magnesium, and sodium (0.1, 1.0, 10, 100 and 1000 ppm) and it was found that calcium affected the binding of platinum(II and IV) to the alfalfa biomass. It was determined that magnesium and sodium did not interfere appreciably with the binding of platinum in either of the oxidation states studied. Finally, batch experiments were performed with Mg²⁺, Ca²⁺ and Na⁺ in solutions at various concentrations, and it was observed that the binding was affected similarly to that by calcium alone.     

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to −80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution.     

In vivo biodistribution,antioxidant and hemolysis tendency of superparamagnetic iron oxide nanoparticles – Potential anticancer agents

Ferromagnetic and superparamagnetic oxide nanoparticles are of particular attention because of their possible use in various fields ranging from bio-nanotechnology to spintronics. Detailed magnetic, dielectric and impedance investigations are crucial for the above-mentioned applications. This study deals with the exploration of various iron oxide phases under as-synthesized conditions by sol–gel method. pH of the sols is varied in the range of 1 to 11. X-ray diffraction (XRD) analysis indicate amorphous behavior for nanoparticles synthesized using pH 1 and 3. Nanoparticles synthesized using pH 2 and 4–6 exhibit hematite phase of iron oxide. Whereas structural transition to maghemite phase is observed for pH 7–8. Nanoparticles synthesized using high pH values, i.e. 9–11, exhibit structural transition towards magnetite phase of iron oxide. Hematite nanoparticles exhibit superparamagnetic and ferromagnetic hysteresis curves with saturation magnetization of ～ 24 emu/g and ～ 13–17 emu/g at pH 2 and pH 4–6, respectively. Maghemite nanoparticles exhibit superparamagnetic (pH 7) and ferromagnetic (pH 8) response with saturation magnetization of ～ 69 and ～ 42 emu/g, respectively. Fe₃O₄ nanoparticles exhibit superparamagnetic (pH 9–10) and ferromagnetic (pH 11) behavior with saturation magnetization of ～ 88, 87 and 52 emu/g, respectively. High grain boundary resistance contributed towards high dielectric constant of ～ 99, 109 and 154 (log f = 5.0) at pH 2, 7 and 9. Detailed impedance values indicate dominant role of grain boundaries in the conductivity of iron oxide nanoparticles. Superparamagnetic iron oxide (pH 9) exhibits strong antioxidant activity along with a very weak hemolytic response. The findings of cell lysis reveal that synthesized nanoparticles have a potential to combat dangerous cancer cells. Drug efficacy results show that after 120 min the encapsulation efficacy reaches a peak of ～ 83 % using curcumin, a naturally existing drug. In vivo biodistribution of nanoparticles was studied in Rabbit model. Synthesized nanoparticles are labelled using Technetium-99 m. Whereas, labeling efficacy and stability was examined using =nstant thin layer chromatography (ITLC) process. In vitro and in vivo results suggest potential anti-cancer applications of as-synthesized superparamagnetic nanoparticles.