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Integration and storage of bioactive reagents is an important and challenging task in microfluidic paper-based analytical devices (μPADs). Here, we developed a convenient and universal method to store proteins and preserve their activities in μPADs by using aqueous two-phase systems (ATPs) evolved film. A polyethylene glycol (PEG)-dextran (DEX) double-layer film was formed through dehydration of ATPs. Functional biomolecules were stored in the bottom DEX layer on the basis of the biased partitioning and rehydrated conveniently by simple addition of buffer solution at usage. As a demonstration, enzyme immunoassay (EIA) of carcinoembryonic antigen was performed successfully on μPAD integrated with antibodies. Even after 104 days of storage at 4 °C and ambient conditions, the EIA signal just lost less than 10% and 30%, which meet the storage requirements of invitro diagnosis reagents. The ATPs evolved double-layer film has double functions of stabilization and insulation, and provide a high efficiency of biomolecule preservation, thereby promoting the applications of μPADs in POC diagnostic assay.

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2–3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

Made available in DSpace on 2018-12-11T16:45:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-08 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This paper describes the fabrication of 2D and 3D microfluidic paper-based analytical devices (μPADs) for monitoring glucose, total protein, and nitrite in blood serum and artificial urine. A new method of cutting and sealing filter paper to construct μPADs was demonstrated. Using an inexpensive home cutter printer soft cellulose-based filter paper was easily and precisely cut to produce pattern hydrophilic microchannels. 2D and 3D μPADs were designed with three detection zones each for the colorimetric detection of the analytes. A small volume of samples was added to the μPADs, which was photographed after 15 min using a digital camera. Both μPADs presented an excellent analytical performance for all analytes. The 2D device was applied in artificial urine samples and reached limits of detection (LODs) of 0.54 mM, 5.19 μM, and 2.34 μM for glucose, protein, and nitrite, respectively. The corresponding LODs of the 3D device applied for detecting the same analytes in artificial blood serum were 0.44 mM, 1.26 μM, and 4.35 μM. Departamento de Química Universidade Federal de São Carlos Intituto de Química-UNESP, Av. Francisco Degni s/n Intituto de Química-UNESP, Av. Francisco Degni s/n CNPq: 140484/2013-2 FAPESP: 2015/19890-1 CNPq: 427727/2016-2

Microfluidic paper-based analytical devices (μPADs) for molecular detection have great potential in the field of point-of-care diagnostics. Currently, a critical problem being faced by μPADs is improving their detection sensitivity. Various preconcentration processes have been developed, but they still have complicated structures and fabrication processes to integrate into μPADs. To address this issue, we have developed a novel paper-based preconcentrator utilizing ion concentration polarization (ICP) with minimal addition on lateral-flow paper. The cation selective membrane (i.e., Nafion) is patterned on adhesive tape, and this tape is then attached to paper-based channels. When an electric field is applied across the Nafion, ICP is initiated to preconcentrate the biomolecules in the paper channel. Departing from previous paper-based preconcentrators, we maintain steady lateral fluid flow with the separated Nafion layer; as a result, fluorescent dyes and proteins (FITC–albumin and bovine serum albumin) are continuously delivered to the preconcentration zone, achieving high preconcentration performance up to 1000-fold. In addition, we demonstrate that the Nafion-patterned tape can be integrated with various geometries (multiplexed preconcentrator) and platforms (string and polymer microfluidic channel). This work would facilitate integration of various ICP devices, including preconcentrators, pH/concentration modulators, and micro mixers, with steady lateral flows in paper-based platforms.

Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp’s susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5–15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30–40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

In this report, we describe a simple, low-cost, straight forward and highly reproducible fabrication method of microfluidic systems. This system was cut on a glass fiber membrane by a common cutter without using any other sophisticated equipment or organic solvents. This format represents a novel type of paper-based microfluidics with high resolution of the microchannel down to ~137 μm, comparable to those made by conventional photolithography. We successfully applied this method to microfluidics to create a star micro-array format of multiplexed urine tests in this study.

BACKGROUND: The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). METHODS AND RESULTS: The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. CONCLUSION: This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

The purpose of this study was to assess the recovery of certain proteins from paper strips used for sampling of gingival crevicular fluid (GCF). During a period of 60 min, proteins were eluent in isotonic phosphate-buffered saline, pH 7.4, without detergent. This eluent did not lyse neutrophils applied to the strips, which might otherwise contribute intracellular substances-, e.g., elastase to the GCF samples. The proteins applied (MW 12-725 kD) were satisfactorily recovered (about 90%). However, free neutrophil elastase, unlike elastase complexed with alpha-2-macroglobulin, could not be recovered from the paper strips. Strips made of polyvinylidene difluoride (Durapore) were also tested and 31% of the applied pure elastase was recovered from them. A comparison of measurements made with a Periotron 6000 on Periopaper and Durapore strips, showed that the values obtained with Durapore were too low for accurate measurements of small volumes of fluid. On the whole, when the elution was made in isotonic buffers at neutral pH and without detergents, the recovery of most proteins was satisfactory and reproducible, and independent of MW in the tested range. However, the study also showed poor recovery of uncomplexed elastase.

Novel methods are demonstrated that enable controlled spatial and temporal rehydration of dried reagents in a porous matrix. These methods can be used in paper-based microfluidic assays to define reagent concentrations over time at zones downstream for improved performance, and can reduce costs by simplifying the manufacturing process with the use of a single porous substrate. First, the creation of uniform reagent pulses from patterned arrays of dried reagent is demonstrated. Second, reagents are stored dry in separate regions of the porous matrix so that they can be combined upon rehydration for immediate use in the device. Third, reagents are reconstituted sequentially from dry storage depots with tunable delivery times. Fourth, the total time for dissolution is varied to achieve a range of reagent delivery times to a downstream region. Finally, the utility of these control methods is demonstrated in the context of real-time reagent rehydration and mixing on a porous device.

The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

Synthetic peptides have been used to define specificity determinants and to distinguish reactivities of numerous protein kinases and phosphoprotein phosphatases. Direct analysis of peptide phosphorylation is most often determined using P81 phosphocellulose paper to separate modified peptide and unreacted [gamma-32P]ATP; however phosphopeptide dephosphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase assay for the determination of phosphopeptide dephosphorylation. The S6-21 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphosphorylated carboxyl terminal domain of the S6 ribosomal protein, was phosphorylated by a human placenta S6 kinase and dephosphorylation by purified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new assay. Results comparable to those obtained with the ion-exchange chromatography were obtained, and the assay was significantly less expensive, more rapid, and more accurate than methods previously used to quantitate phosphopeptide dephosphorylation.

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based "stamp") as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimetre. This method provides a new tool to pattern biochemicals-reagents, antigens, proteins, and DNA-on planar substrates. The accuracy of the volume of fluid delivered in simple paper-to-paper printing is low, and although the pattern transfer is rapid, it is better suited for qualitative than accurate, quantitative work. By patterning the paper to which the transfer occurs using wax printing or an equivalent technique, accuracy increases substantially.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed ‘Key paper’ is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

The preparation and use of prepunched and preprinted sheets of filter paper disks for radioassay of protein synthesis and other reactions is described. While similar results are obtained using precut paper circles, the use of a sheet of disks greatly expedites the process and simplifies the handling of numerous samples.

A method is described for determination of protein in biological preparations containing various interfering substances normally present in extraction media. The main steps of the procedure consist in depositing the protein solution on filter paper strips and removing all small molecular weight substances by washing with a number of aqueous and nonaqueous solvents. The protein remaining on the paper is then determined by a modification of Lowry's colorimetric procedure. The method also permits the determination of protein in radioactive samples which have been previously counted using a liquid scintillation mixture such as Apuasol or toluene-POPOP.

A novel two-dimensional cyanuric chloride-activated (CCA) paper has been developed. It is composed of a cellulosic base, covalently bound cyanuric chloride, and microprecipitated complex cyanuric chloride-sodium chloride crystals on its surface. CCA paper covalently binds nucleic acids and proteins. Its binding capacity for nucleic acids is about 400 micrograms/cm2. Sealed into nitrogen-filled bags and stored at -20 degrees C, it retains its binding activity for at least a year and is always ready for use. CCA paper has been successfully used for capillary and electroblotting of DNA, RNA, and proteins (Southern, Northern, and Western blotting) as well as for dot tests. Furthermore, it was applied to colony and plaque hybridization. A unique property of it is that it permits the staining of proteins after blotting and subsequent performance of radioimmunological detection of specific protein components. This has proven advantageous in two-dimensional Western blotting experiments. Of further importance is its ability to bind DNA fragments from one up to several hundred bp from polyacrylamide sequencing gels.

We describe a method by which complex protein mixtures are fractionated by standard one-dimensional Na-DodSO4/polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrophoresis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]-methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins could be detected by sequential treatment of diazobenzyloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 125I-labeled Staphyloccus aureus protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody, By one-dimensional gel transfer/immunoautoradiography, seven virion proteins were detected as prominent bands and several others as weaker bands. By two-dimensional gel transfer/immunoautoradiography, several additional viral proteins were detected. By use of anti-DNA binding protein serum, the Mr 73,000 protein and Mr 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.

The detectability by India ink staining of proteins and peptides dot-blotted on nitrocellulose paper was assessed before and after fixation. Fixation considerably increased the detectability of proteins and peptides. Denaturation by KOH treatment or baking at 100 degrees C for 15 min gave the best results. Precipitation by isopropanol/acetic acid gave intermediate results, whereas crosslinking with glutaraldehyde improved the detectability of small peptides, but not of proteins. Ferridye and Aurodye were also tested after baking. Both dyes were more sensitive and stained more proteins and peptides than India ink. In all cases the detectability of peptides smaller than Mr 1500 was poor.

Allergenic proteins were extracted from one silk batch that was imported to be used as filling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides.

Nerve ending particles isolated from prenatal and postnatal rats by means of density-gradient centrifugation in a ficoll medium actively incorporated [32P]-orthophosphate into nucleotides, phosphoproteins and phospholipids. This process requires Mg2+; is dependent on malate plus pyruvate but not glucose, and is inhibited by dinitrophenol, gramicidin, oligomycin, azide, p-chloromercuribenzoate, CaCl2 but not iodoacetate. It was concluded that the metabolic energy at the synapse is derived largely from the mitochondria of the synaptic complex rather than from its cytoplasm. Thin-layer chromatography and paper electrophoresis revealed that the metabolically active phospholipids, namely phosphatidic acid, phosphatidyl inositol and the polyphosphoinositides which were found to be tightly bound to the phosphoprotein fraction, contained more than 90% of the total radioactivity but constituted less than 9% of the total phospholipids. The highest phosphoprotein and phospholipid content of the nerve endings (expressed in μmoles P/mg nerve ending protein) as well as maximal 32P-incorporation occurred just prior to and continued into the stage when functional changes are formed. It is suggested that an increase in the amount of enzymatic activity coupled with an increase in the permeability of the synaptosomal membrane to inorganic phosphate and other metabolites could trigger the rapid morpho-biochemical and functional changes observed in rat brain during this period of development. A simple density-gradient withdrawing device for the isolation of the submitochondrial particles after density-gradient centrifugation of the conventional mitochondrial fraction was described.

1 A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ɛ-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2 The technique has been successfully tested with insulin. 3 When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4 A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

1. 1. Four proteins of known sequence (lysozyme, myoglobin, ribonuclease and insulin) have been treated with NaOH and ammonia solutions, and the amounts of lysinoalanine and β-aminoalanine estimated in the hydrolysates by high-voltage paper electrophoresis. The relative amounts of each amino acid are explained by reference to the positions of cystine and serine residues in relation to lysine residues and amide side-chains in the proteins. 2. 2. The evidence indicates that β-aminoalanine is formed by direct addition of ammonia to α-aminoacrylic acid residues, and not by initial formation of a cross-link between α-aminoacrylic acid and asparagine or glutamine residues. 3. 3. Further evidence has been found for the formation of α-aminoacrylic acid from residues other than cystine, by treatment of myoglobin with alkali. 4. 4. Treatment of β-lactoglobulin and wheat gluten, whose sequences are unknown, has enabled tentative suggestions to be made about the relative positions of certain residues in these proteins.

We examine the effects of filter efficiency on DNA-protein binding data obtained by the popular nitrocellulose filter-binding assay. Graphical procedures for determining the efficiency parameter epsilon (for the efficiency of retention of DNA on the filter, per bound protein molecule) are established. Filter efficiency modified formulas for determining thermodynamic binding parameters are derived for four simple prototypes of DNA-protein binding systems. The effects of experimental error on discrimination between models are considered. Finally, we discuss conceptual errors often made in calculating binding stoichiometry from filter-binding experiments and suggest a general protocol for the analysis of filter-binding data.

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in situ with pyridoxal 5′-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5′-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse ((ab′) 2 antibody or a peroxidase-linked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices.

The possibility of complex formation between chloroquine and hyaluronate-protein has been investigated by a spectrophotomefric method. Interaction was observed between chloroquine and a hyaluronate-protein containing 3.4% protein, but no interaction occurred between chloroquine and hyaluronate-protein preparations containing 2.36 and 2.30% protein, respectively. The protein in two preparations appears to be tightly bound to the hyaluronate. The larger amount of protein in one of the preparations appears to be responsible for the binding process.

Zygorhynchus vuilleminii , a nonmelanin-containing fungus, was not lysed by mycolytic actinomycetes. Several enzymes and Streptomyces enzyme preparations digesting walls of other fungi were without appreciable activity on walls of Zygorhynchus species. A bacterium able to solubilize a portion of the Zygorhynchus wall released little or no reducing sugars from these structures. Fractions of Z. vuilleminii walls were resistant to glucanase hydrolysis, but certain fractions were digested by chitinase and microbial enzyme preparations. The walls and several wall fractions were not readily susceptible to degradation by a soil community. Walls of lysis-resistant Zygorhynchus species contained glucosamine, fucose, glucuronic acid, and galactose but little or no glucose. Resistant wall fractions were rich in uronic acid and fucose, whereas the readily degradable fractions contained abundant glucosamine. Cultural conditions affected the extent of digestion and composition of the walls. Possible reasons for the resistance of Zygorhynchus to lysis in nature are discussed.

Duodenal fluids from cystic fibrosis and control subjects were analyzed for proteins and mucoproteins by quantitative paper-strip electrophoresis. Prominent quantitative differences were found among the nine major proteins and mucoproteins of the two subject groups. Three proteins and two mucoproteins did not differ between the groups, but the other two proteins and two mucoproteins did differ. The cystic fibrosis subjects had: (I) a relative excess of substances designated as mucoprotein O and protein C5; and (2) a relative deficiency of mucoprotein C4 and protein O. It is notable that cystic fibrosis is characterized by a quantitative difference pattern rather than by a qualitative difference.

Carboxymethyl cellulose chromatography permits separation of five distinct toxic proteins from the venom of the Indian cobra ( Naja naja ). These account for 90 per cent of the toxicity against mice. After gel filtration on Sephadex G-50 columns, three of the toxic fractions are more potent than the crude venom by factors of 6–9. They differ from one another in their sensitivities to heat and trypsin inactivation. Phospholipase does not appear to contribute significantly to the lethal effect. The two most abundant fractions, cobramines A and B, are less toxic than the crude venom and account for its cardiotoxic activity.