Purification of nerve growth factor from the venom of the Central Asian cobraNaja oxiana

A highly purified electrophoreticaly homogeneous protein with a NGF activity of 10·10⁵ BU/mg of protein have been isolated from the venom of the Central Asian cobra by gel-filtration and ion-exchange chromatography followed by preparative isolectric focusing in a thin layer of Sephadex. It has been shown that the NGF isolated is characterized by a molecular weight in the range of 20–30 kD and a pI value of about 7.0.     

Stability of immobilized frog epidermis tyrosinase

Purified frog epidermis tyrosinase was immobilized on the following supports: CNBr-Sepharose 4B, Enzacryl AA, Enzacryl AH, Enzacryl Polythiolactone, and Enzacryl Polyacetal. The enzyme was active on all supports except when Enzacryl Polyacetal was used. The stability increased on immobilization. Enzacryl AA was the best support assayed. The Enzacryl AA-enzyme complex was 30- to 40-fold more stable to inactivation reaction than soluble enzyme, and maintained its activity when stored and assayed repeatedly. The immobilized enzyme on the other supports was also more stable than the soluble form. The pH-activity profile, thermal stability, storage stability, and the effect of protein concentration on activity of the immobilized enzyme have been studied. The properties observed for the immobilized enzyme were different than those of the soluble enzyme. The main reason for this difference could be due to enzyme modification through tyrosine groups of the enzyme; to conformational changes produced in the union to the matrix; and to microenvironmental differences created by the matrix.     

Chemiluminescence microarrays in analytical chemistry: a critical review

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review. Figure

Achievements in the development of CL microarray analysis platforms     

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (¹⁰Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods. Figure

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A study of the hydration of ribonuclease A using isothermal calorimetry

This work is part of a systematic study undertaken to find the excess thermodynamic functions of binary protein–water systems. Isothermal calorimetry and water sorption measurements were applied to characterize the hydration dependencies of the excess thermodynamic functions. The advantages of our methodology are (i) we are able to simultaneously determine the excess partial quantities of water and proteins; (ii) these thermodynamic quantities can be determined in the entire range of water content. Here, in particular, the excess partial enthalpies of water and bovine pancreatic ribonuclease A (RNase A) have been determined. The excess partial enthalpies for RNase A are compared with the published data for several unrelated globular proteins (lysozyme, chymotrypsinogen A, serum albumin, lactoglobulin). These biomacromolecules represent a series of proteins in which the hydrophobicity of proteins is gradually changed in a wide range. It was found that the excess partial quantities for the studied proteins are determined by the hydration of the hydrophilic and hydrophobic protein groups. The more hydrophilic a protein, the more significant a hydrophilic hydration contribution is and vice versa. RNase A is the most hydrophilic protein under the study. This protein has the most significant hydrophilic hydration contribution. Lactoglobulin is the most hydrophobic protein under the study. This protein has the most significant hydrophobic hydration contribution.     

Sepharose-immobilized triazine dyes as adsorbants for human lymphoblastoid interferon purification

An extensive selection of immobilized triazine dyes have been examined for their potential as adsorbants for human lymphobalstoid interferon. Procion red HE7B was selected as the most suitable for preparative scale purification. Sepharose-immobilized procion red HE7B is able to bind 10⁵ reference units/mL of interferon from cell supernatants and can be eluted with at least 25-fold purification and 90% yield by a KC1 gradient. Further purification was obtained either by reapplying the eluted interferon after dialysis to the dye column or by gel filtration on Ultrogel AcA 34 after lyophilization and dialysis. The latter procedure gave a final activity of about 10⁶ U/mg protein and approximately 75% recovery of interferon activity.     

Geopolymers

Spectacular technological progress has been made in the last few years through the development of new materials such as ‘geopolymers’, and new techniques, such as ’sol-gel’. New state-of-the-art materials designed with the help of geopolymerization reactions are opening up new applications and procedures and transforming ideas that have been taken for granted in inorganic chemistry. High temperature techniques are no longer necessary to obtain materials which are ceramic-like in their structures and properties. These materials can polycondense just like organic polymers, at temperatures lower than 100?. Geopolymerization involves the chemical reaction of alumino-silicate oxides (Al³⁺ in IV-fold coordination) with alkali polysilicates yielding polymeric Si-O-Al bonds; the amorphous to semi-crystalline three dimensional silico-aluminate structures are of the Poly(sialate) type (-SiO-Al-O-), the Poly(sialate-siloxo) type (-Si-O-Al-O-Si-O-), the Poly(sialate-disiloxo) type (-Si-O-Al-O-Si-O-Si-O-). This new generation of materials, whether used pure, with fillers or reinforced, is already finding applications in all fields of industry. Some examples:

pure: for storing toxic chemical or radioactive waste, etc.

filled: for the manufacture of special concretes, molds for molding thermoplastics, etc.

reinforced: for the manufacture of molds, tooling, in aluminum alloy foundries and metallurgy, etc.

These applications are to be found in the automobile and aerospace industries, non-ferrous foundries and metallurgy, civil engineering, plastics industries, etc.     

Immunocapillarymigration

The method is based upon the attachment of antibodies to a porous insoluble support and the subsequent capillary migration of the antigen-containing solution in the support. The antigen-covered area thereby obtained was visualized with fluoresceinor horseradish peroxidase-labeled antibodies. The height of this area increased with increasing antigen concentration. The method was used to quantitate C-reactive protein.     

Establishing analytical comparability for “biosimilars”: filgrastim as a case study

Biosimilars are defined as biotherapeutic drugs that have been shown to have comparable quality, safety, and efficacy to the original product. Fuelled by the patent cliff in the next 5 years, the focus of the biopharmaceutical industry is gradually shifting towards production of biosimilars. Scientific and regulatory issues around development and approval of these biosimilars have been a topic of great interest and debate recently. Unlike the conventional small molecular weight drugs, biosimilars exhibit high complexity at the molecular level. Slight variations during the manufacturing of these complex protein molecules may lead to the significant changes in the safety and efficacy profile of the therapeutic product. Establishing comparability to the reference product is essential for successful approval of a biosimilar product. Analytical comparability provides the foundation to this exercise. This paper presents data from such an exercise involving use of several orthogonal analytical tools for establishing analytical comparability. Granulocyte colony-stimulating factor (GCSF/Filgrastim) expressed in Escherichia coli has been selected as the model protein. The approach would be of interest to those engaged in development and commercialization of biosimilars. Figure

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The IUPAC aqueous and non-aqueous experimental pKa data repositories of organic acids and bases

Accurate and well-curated experimental pKa data of organic acids and bases in both aqueous and non-aqueous media are invaluable in many areas of chemical research, including pharmaceutical, agrochemical, specialty chemical and property prediction research. In pharmaceutical research, pKa data are relevant in ligand design, protein binding, absorption, distribution, metabolism, elimination as well as solubility and dissolution rate. The pKa data compilations of the International Union of Pure and Applied Chemistry, originally in book form, have been carefully converted into computer-readable form, with value being added in the process, in the form of ionisation assignments and tautomer enumeration. These compilations offer a broad range of chemistry in both aqueous and non-aqueous media and the experimental conditions and original reference for all pKa determinations are supplied. The statistics for these compilations are presented and the utility of the computer-readable form of these compilations is examined in comparison to other pKa compilations. Finally, information is provided about how to access these databases.     

Evaluation of stability of silica-supported Fe–Pd and Fe–Pt nanoparticles in aerobic conditions using thermal analysis

The applications of zerovalent iron nanoparticles (nZVI) exploit their high reactivity which decreases due to oxidation in aerobic conditions during manufacture, application, and storage. In this study, we present the new procedure for estimation of the nZVI stability to oxidation in air. The procedure is suitable for characterization of the novel materials based on the supported nZVI. Nanoscale particles were synthesized inside porous silica supports by incipient wetness impregnation with the metal precursor solutions followed by thermal treatment. The TG–DTA studies revealed the decomposition temperature of the supported precursors, as well as the interaction of Fe and precious metal precursors, which resulted in the formation of alloy nanoparticles. Characterization of the samples by XRD confirmed the formation of the nanoparticles of the metallic Pd, Pt, and Fe phases supported on SiO₂ carriers, as well as the formation of solid solutions based on the structure of precious metals. The new procedure for estimation of the nZVI stability included (1) TPR with hydrogen up to 400–425 °C followed by isothermal reduction at these temperatures; (2) in situ reoxidation with oxygen at room temperature. The samples were reduced “as obtained” and after in situ reoxidation. The results of the TPR studies exhibited that introduction of both Pd and Pt protected the Fe nanoparticles from oxidation with oxygen and air at ambient conditions.     

The Effect of Arg on the Structure Perturbation and Chaperone Activity of α-Crystallin in the Presence of the Crowding Agent,Dextran

α-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses. It has a molecular weight of 20 kDa and is composed of two subunits: αA and αB. α-Crystallin is a member of the small heat shock protein (sHsps) family that has been shown to prevent protein aggregation. Small molecules are organic compounds that have low molecular weight (<800 Da). Arginin (Arg) is a small molecule and has been shown to prevent protein aggregation through interaction with partially folded intermediates. In this study, the effect of Arg on the chaperone activity of α-crystallin in the presence of dextran, as a crowding agent, against ordered and disordered aggregation of different target proteins (α-lactalbumin, ovotransferrin, and catalase) has been investigated. The experiments were done using visible absorption spectroscopy, ThT-binding assay, fluorescence spectroscopy, and CD spectroscopy. The results showed that in amorphous aggregation and amyloid fibril formation, both in the presence and absence of dextran, Arg had a positive effect on the chaperone action of α-crystallin. However, in the presence of dextran, the effect of Arg on the chaperone ability of α-crystallin was less than in its absence. Thus, our result suggests that crowding interior media decreases the positive effect of Arg on the chaperone ability of α-crystallin. This is a very important issue, since we are trying to find a mechanism to protect living cells against the toxic effect of protein aggregation.     

Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry

Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H₂ or -D₂. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H₂ and formaldehyde-D₂, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R ² of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.     

Characterization of chitosan microparticles reinforced cellulose biocomposite sponges regenerated from ionic liquid

The chitosan-microparticles reinforced cellulose biocomposite sponges regenerated from ionic liquid were prepared and characterized. Fourier transform infrared (FTIR) spectroscopy confirmed that the cellulose dissolved in 1-allyl-3-methylimidazolium chloride without derivatization. Chitosan particles as reinforcement were incorporated into the cellulose matrix. FTIR spectra indicated hydrogen bonding between hydroxyl groups of cellulose and chitosan. The biocomposite sponges showed uniform three-dimensional interconnected porous structures. The breaking strength of the sponges increased significantly, from 0.09 to 0.32 MPa with the addition of 1.0 wt% chitosan. The sponges also demonstrated excellent antibacterial activity against S. aureus and E. coli with the average inhibition zone diameters >2 mm and the inhibition rate higher than 80 %. Furthermore, the biocomposite sponges exhibited good moisture penetrability and high porosity. The water uptake ability of the sponge was >25 times of its weight in water with a fast swelling. The chitosan/cellulose composite sponge is expected to be a promising material for potential applications as wound dressing.     

Purine nucleoside phosphorylase: Immobilization by covalent chromatography and a study on its sulfhydryl groups

Covalent chromatography is used for studying the role of sulfhydryl groups in pig brain purine nucleoside phosphorylase (PNP). This enzyme has been immobilized on an insoluble polymeric reagent (thiol-Sephnrose 4B) by a thiol-disulfide interchange reaction between the disulfide groups of the gel and some of its thiol groups. The immobilized enzyme retained its activity and the coupling was reversible under reducing conditions, allowing the recovery of the enzymic activity. These results suggest that PNP contains nonessential sulfhydryl groups that can react with the thiol-Sepharose. On the other hand, inactivation with some thiol reagents shows that thiol groups directly involved in the catalytic activity are present at or near the active site. The technique described should be generally useful in the immobilization of thiol-containing proteins and in the characterization of these thiol groups.     

Qualitative analysis of molecular structure of polypropylene fibres on the basis of X-ray diffraction patterns

The present paper reports an investigation in which X-ray diffraction (WAXS and SAXS) patterns are applied to obtain qualitative information regarding supermolecular structure of drawn und undrawn samples of polypropylene fibres. The tensile properties, increase of long period, formation of microvoids and lamellar orientation upon drawing of the fibre have been brought into focus.     

Porous liquids – the future is looking emptier

The development of microporosity in the liquid state is leading to an inherent change in the way we approach applications of functional porosity, potentially allowing access to new processes by exploiting the fluidity of these new materials. By engineering permanent porosity into a liquid, over the transient intermolecular porosity in all liquids, it is possible to design and form a porous liquid. Since the concept was proposed in 2007, and the first examples realised in 2015, the field has seen rapid advances among the types and numbers of porous liquids developed, our understanding of the structure and properties, as well as improvements in gas uptake and molecular separations. However, despite these recent advances, the field is still young, and with only a few applications reported to date, the potential that porous liquids have to transform the field of microporous materials remains largely untapped. In this review, we will explore the theory and conception of porous liquids and cover major advances in the area, key experimental characterisation techniques and computational approaches that have been employed to understand these systems, and summarise the investigated applications of porous liquids that have been presented to date. We also outline an emerging discovery workflow with recommendations for the characterisation required at each stage to both confirm permanent porosity and fully understand the physical properties of the porous liquid.

The realisation of permanent microporosity in liquids transforms the way functional porosity may be implemented. Considering recent advances, we explore the developing theory of porous liquids and delve into the discovery process and applications.     

Immunosorbents: A new tool for pesticide sample handling in environmental analysis

 Immunoaffinity techniques have been widely used for the determination of different analytes in the medical field. However the use of antibodies immobilized in an appropriate support material to preconcentrate pesticides from environmental samples is only recent. The production of antibodies, election of supports, antibody immobilization procedures, elution of analytes from immunosorbents and the more recent applications in the field of pesticide analysis are reviewed. The present review concludes that immunosorbents have great potential and discusses the present limitations and expected future trends. Received: 29 July 1996 / Accepted: 14 August 1996     

Routine quality control of11C-labelled radiopharmaceuticals by high pressure liquid chromatography

Radio high pressure liquid chromatography (radio-HPLC) is the method of choice for quality control of radiopharmaceuticals labelled with short lived isotopes. Our preparations of “no carrier added”¹¹C-labelled palmitic acid and L-methionine are both designed to end with a HPLC separation on either silica gel or C-18 reversed phase material. Since the crude reaction mixtures contain milligram amounts of inactive substrate materials, both separations must be carried out at preparative scale. Nevertheless they are performed in less than 10 min. The most critical factor for the separation of¹¹C-palmitic acid from the main by-product pentadecane is the solvent composition: while the¹¹C-L-methionine separation is especially sensitive to pH variations.     

In Situ Characterization of Proteins Using Laserspray Ionization on a High-Performance MALDI-LTQ-Orbitrap Mass Spectrometer

The MALDI-LTQ-Orbitrap XL mass spectrometer is a high performance instrument capable of high resolution and accurate mass (HRAM) measurements. The maximum m/z of 4000 precludes the MALDI analysis of proteins without generating multiply charged ions. Herein, we present the study of HRAM laserspray ionization mass spectrometry (MS) with MS/MS and MS imaging capabilities using 2-nitrophloroglucinol (2-NPG) as matrix on a MALDI-LTQ-Orbitrap XL mass spectrometer. The optimized conditions for multiply charged ion production have been determined and applied to tissue profiling and imaging. Biomolecules as large as 15 kDa have been detected with up to five positive charges at 100 K mass resolution (at m/z 400). More importantly, MS/MS and protein identification on multiply charged precursor ions from both standards and tissue samples have been achieved for the first time with an intermediate-pressure source. The initial results reported in this study highlight potential utilities of laserspray ionization MS analysis for simultaneous in situ protein identification, visualization, and characterization from complex tissue samples on a commercially available HRAM MALDI MS system. Graphical Abstract

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