bluuebunny/crossref_metadata_2025 huggingface dataset:
Low temperature, aerosol‐assisted chemical vapor deposition (AACVD) of CdS, ZnS, and Cd1‐xZnxS using monomeric single‐source precursors: M(SOCCH3)2 TMEDA
Authors: Nyman May , Hampden‐Smith Mark J. , Duesler Eileen N.
Published: May 2005

Novel, monomeric single‐source precursors for the CVD of metal sulfides, which are widely used in solar cell technology, are described. The Figure shows the molecular structure of M(SOCCH3)TMEDA, where TMEDA = N,N,N,N‐tetramethylethylenediamine, for the Cd and Zn complexes. These compounds allow the lowest deposition temperatures so far reported for highly oriented crystalline, stoichiometric films.

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Preparation of copper sulfide thin layers by a single‐source MOCVD process
Authors: Nomura Ryôki , Miyawaki Keico , Toyosaki Takayuki , Matsuda Haruo
Published: May 2005

Metal dithiocarbamate complexes are also potential candidates for the CVD of metal sulfides (see previous article). This paper reports on an investigation by mass fragmentation initiated by electron impact of the vapor phase decomposition of a copper complex in order to clarify the decomposition route and to assess its suitability as a single‐source precursor for the MOCVD growth of cuprous sulfide. Reproducible growth of crystalline layers was found and the results suggested a two‐step growth mode.
Voltammetric monitoring of photochemical reactions: The photosubstitution of group VIA hexacarbonyls
Authors: Compton R. G. , Barghout R. , Eklund J. C. , Fisher A. C.
Published: May 2005

Voltammetry at an irradiated platinum channel electrode is used to monitor photosubstitution reactions of the Group VIA hexacarbonyls, M(CO)6 (M = Cr,Mo,W), in acetonitrile solution, in which up to three CO ligands are replaced by solvent molecules. It is shown that this methodological approach can be used to suggest reaction mechanisms and to quantify rates of photoreactions, so giving estimates of quantum yields.
Determination of dimethoate in water by cathodic stripping square‐wave voltammetry
Authors: Hernández P. , Ballesteros Y. , Galán F. , Hernández L.
Published: May 2005

This article describes the direct‐determination of the pesticide dimethoate In river water in an alkalin media and accumulaton medium through cathodic stripping voltammetry. By employing an alkaline potential of 30 mV, reduction peak appears at −0.7 V. The detection limit is 1 ng ml−1.
Anodic behavior of some Vinca alkaloids with cytostatic activity: Effect of pH
Authors: Brett A. M. Oliveira , Grazina M. M. M. , Macedo T. R. A. , Raimundo D.
Published: May 2005

The anodic oxidation mechanism of the Vinca alkaloids vinblastine, its semisynthetic derivatives vindesine and 5′‐noranhydrovinblastine, and of vincristine, all with closely similar structures, was studied at a glassy carbon disk electrode in buffered aqueous media using differential pulse and cyclic voltammetry. The effect of pH on the mecharism showed that it is a complex multistep electron transfer with deprotonation steps involved and that the final products, dimers or polymers, adsorb strongly to the electrode, forming an unreactve film on the electrode surface. The electrochemical differential pulse voltammograms showed a correlation between the vinblastine derivatives, whereas vincristine reacts at different potentials. These differences in the anodic oxidation mechanisms of the compounds studied can be related to their neurotoxicity and myelosuppression effects. These effects may be due to the metabolite and/or degradation products of the compounds and result from how they are attached to, or transported across, cellular structures such as cell membranes.
Rapid coulometry of tocopherols in ethanol and chloroform using ferricyanide ion mediator
Authors: Uchiyama Shunichi , Kurokawa Yuichi , Hasebe Yasushi , Suzuki Shuichi
Published: May 2005

In order to estimate the absolute determination method of tocopherols, rapid coulometry has been carried out by using a vapor grown carbon fiber electrode impregnated with ethanol‐water solution containing ferricyanide ion as a mediator. The electrolysis time of 1 × l0−3 M α‐tocopherol in ethanol was below 30 seconds, and its current efficiency was attained to be 100.1% when an ethanol content in pH 4.0 electrolyte is 20%. Four isomers of vitamin E in chloroform were also determined by indirect coulometry in which ferricyanide solution was mixed with sample solution for 10 minutes prior to electrolysis and the current efficiencies of α‐, β‐, γ‐, and δ‐tocopherols were obtained to be 97.6, 100.5, 89.7, and 83.7%, respectively, and the relative standard deviations were below 1%.
Electrochemical oxidation of thiobenzanilide
Authors: Gabriel Jiří , Němec Ivan , Zimová Naďa , Hanuš Vladimír , Waisser Karel
Published: May 2005

The electrochemical oxidation of thiobenzanilide was studied in anhydrous acetonitrile as a model for the enzymatic oxidation of the substance. It has been found that a single electron is exchanged in the presence of 5 × 10−2 M HClO4 The oxidation products were identified as H2S, benzanilide, and 2‐phenylbenzthiazole.
The effect of the nature of the polymer backbone on the stability and the analytical response of polymer‐modified electrodes
Authors: Doherty A. P. , Stanley M. A. , Arana G. , Koning C. E. , Brinkhuis R. H. G. , Vos J. G.
Published: May 2005

The synthesis, characterization, and sensor application of the novel redox polymer [Os(bipy)2(PS)7.5(DMAP)2.5Cl]Cl, where bipy = 2,2′‐bipyridyl, PS = polystyrene, and DMAP = poly[4‐(N‐methyl‐N‐p‐vinylbenzylamino) pyridine], are described. The charge transport properties of electrodes modified with the redox material are investigated using cyclic voltammetry and chronoamperometry. The modified electrode behaves as an efficient electrocatalyst for the outer‐sphere reduction of FeIII, with the cross‐exchange reaction occurring at the surface of the polymer (Sk″) at concentrations less than 1.0 × 10−3 mol dm−3 FeIII, with a change over to the Ste, kinetic regime at higher substrate concentrations. Direct agreement was observed between the kinetic behavior at rotating disk electrodes and in thin‐layer flow cells. Application of the modified electrodes for the determination of iron in pharmaceutical formulations and the long term stability of the electrodes are investigated. The results obtained are compared with those reported for the analogous metallopolymer [Os(bipy)2(PVP)10Cl]Cl, where PVP is poly‐4‐vinylpyridine.
Differential pulse stripping voltammetric determination of thallium with an 8‐hydroxyquinoline‐modified carbon paste electrode
Authors: Cai Qiantao , Khoo Soo Beng
Published: May 2005

A carbon paste electrode incorporating 8‐hydroxyquinoline was fabricated to selectively accumulate TlI at open circuit. The accumulated TlI, after reduction to metallic thallium at the electrode surface, was anodically stripped by differential pulse voltammetry. Under optimized conditions, TlI concentrations in the range 1.00 × 10−8 M − 1.00 × 10−5 M gave two linear regions due to different controlling factors for accumulation. A detection limit of 4.90 × 10−9 M (1.00 ppb) was found for a 120‐s accumulation. For 10 determinations of TlI at concentrations of 1.00 × 10−6 M and 1.00 × l0−7 M, relative standard deviations of 2.60% and 3.20% were obtained, respectively. Interferences from BiIII, PbII and CuII could be removed by masking with complexing agents such as sodium diethyldithiocarbamate, EDTA, sodium citrate, etc. A method was developed for thallium speciation and analysis, based on the determination of TlI while masking TlIII with EDTA followed by chemical reduction of TlIII to TlI with hydroxylamine hydrochloride. This method was tested using a synthetic sample containing known concentrations of TlI and TlIII.
Rapid determination of manganese in wastewaters and seawater by stripping potentiometry
Authors: Scollary Geoffrey R. , Chen Guo Nan , Cardwell Terence J. , Vincente‐Beckett Victoria A.
Published: May 2005

A rapid stripping potentiometric method for the determination of trace levels of manganese in seawater and industrial waste waters has been developed. The analysis is performed in the presence of oxygen with an analysis time of less than 2.5 min per sample at manganese concentrations lower than 5μg/L. The precision of the method is satisfactory and the results for 5 seawater and waste water samples compare favorably with graphite furnace atomic absorption spectrophotometry.
A study of ethylamine at a gold rotating ring‐disk electrode using pulsed electrochemical detection at the ring
Authors: Dobberpuhl David A. , Johnson Dennis C.
Published: May 2005

Linear scan (cyclic) voltammetry at the disk with simultaneous pulsed electrochemical detection (PED) at the ring of a rotated ring‐disk electrode (RRDE) is demonstrated to be applicable for studies of the complex anodic behavior of ethylamine at gold electrodes in 0.10 M NaOH. The oxidation of ethylamine at the disk occurs during positive scans concomitantly with formation of surface oxide (Au → AuOH → AuO). However, the final oxide‐covered surface (AuO) is inert for further ethylamine oxidation. Data obtained at the RRDE demonstrate that the total ethylamine signal at the disk is composed of simultaneous contributions from: oxidative desorption of ethylamine preadsorbed at the oxide‐free Au surface and oxidation of ethylamine transported to the disk simultaneously with oxide formation. Based on ring‐disk data, preadsorbed ethylamine is estimated to correspond to a fractional surface coverage of 0.7 ± 0.1 monolayer for 10 to 60 μM ethylamine. Of this coverage, ca. 75% corresponds to ethylamine coadsorbed reversibly with OH− and 25% to ethylamine adsorbed irreversibly by a mechanism concluded to be chemisorption.
Evaluation of different carbon electrodes for adsorptive stripping analysis of nucleic acids
Authors: Cai Xiaohua , Rivas Gustavo , Farias Percio A. M. , Shiraishi Haruki , Wang Joseph , Palecek Emil
Published: May 2005

Recently we have shown (see Ref. [8]) that submicrogram amounts of nucleic acids can be measured by potentiometric stripping analysis (PSA) at carbon paste electrodes. In this article we investigate and compare various carbon electrode materials for use in stripping analysis of nucleic acids. Both the interfacial and redox properties of DNA and RNA are strongly dependent upon the nature of the carbon material and the electrode pretreatment step. In view of the high solvent decomposition background response near the guanine oxidation peak, only potentiometric stripping analysis can be used for convenient quantitation of trace levels (ng/mL) of nucleic acids. The trend in sensitivity: carbon paste > pyrolytic graphite > highly oriented pyrolytic graphite > carbon strip [with detection limits for oligo(dG)20 of 20, 30, 40 and 50 ng/mL, respectively, using 5 min accumulation]. No response was observed for trace measurements at the glassy carbon or carbon fiber electrodes. The influence of the surface pretreatment and accumulation time is described along with the analytical‐performance characteristics at each electrode. Useful information about the adsorption behavior of nucleic acids at different carbon electrodes are also obtained.
A study of the stripping voltammetric behavior of selected metal chelates and its application to automated analysis of river waters
Authors: Maxwell Tracy J. , Franklin Smyth W.
Published: May 2005

The ASV and AdSV behavior of Zn2+, Cd2+, Pb2+, Cu2+, Ni2+ and Sn4+in the presence of selected complexing/chelating agents has been studied. The presence of 2,5‐dimercapto‐l,3,4‐thiadiazole(I) lowers the limit of detection (LOD) for the ASV determination of Zn2+, Cd2+ and Pb2+ in 5 × 10−3 mol dm−3 LiCl supporting electrolyte with a deposition time of 120s to 0.82, 0.17 and 0.34 ppb, respectively, due to participation of the adsorbed complexing agent in the overall process. Similarly, Cd2+ can be determined in the presence of benzimidazole sulfoxides (II) and (III) by ASV in Britton‐Robinson (BR) buffer pH9 with 120s deposition with lower LODs of 0.12 and 0.06 ppb. respectively. AdSV can also be used to determine Cd2+ with (II), (III) and ammonium pyrrolidine dithiocarbamate (IV) with LODs of 0.70, 0.64 and 0.20 ppb, respectively and with Zn2+ having an AdSV LOD of 1.09 ppb using adsorption of its chelate with (IV) at −900mV for 60 s in a supporting electrolyte of 5 × 10−3moldm−3 LiCI. Participation of the adsorbed complexing agent in the ASV process is also observed for Sn4+ and Cu2+ in an oxalate/ammonium chloride/HCl buffer with lowered LODs of 0.51 ppb and 0.76 ppb in the presence of p‐methylene blue (V). The chelating agent 2‐(5‐bromo‐2‐pyridylazo)‐5‐diethylaminophenol(VII) can be used to determine Cd2+,Zn2+ and Pb2+using the AdSV technique down to LODs of 9.3, 2.7 and 6.3 ppb, respectively. Several of these methods have been combined in the development of an automated method for the determination of trace Zn, Cd, Pb and Cu in an artificial river water matrix. The accuracy and precision of this procedure is discussed. Determination of Ni, Sn and As by this automated method and in such a matrix proved to be inaccurate.
Interaction of brilliant cresyl blue and methylene green with DNA studied by spectrophotometric and voltammetric methods
Authors: Liu Junli , Li Jinghong , Dong Shaojun
Published: May 2005

The binding behavior of two cationic dyes, brilliant cresyl blue (BCB) and methylene green (MG) to calf thymus DNA was studied by spectrophotometric and voltammetric methods. A red shift of the adsorption spectra and hypochromism accompany the binding of BCB and MG to calf thymus DNA. In 5 × 10−2 mol dm−3 NaCl, 5 × 10−3 mol dm−3 tris‐ HCl pH 6.87 buffer solution, the apparent binding constants are: KBCB+ 3.0 × 104M−1 (N= 4.13) and K= 8.8 × 104M−1 (n= 4.44). Electrochemical studies show that the formal potentials shift negatively upon addition of DNA, indicating that the oxidized forms of the dyes have stronger affinity to DNA than the reduced ones. K/KBCBH and K/KMGH are evaluated to be 10.39 and 7.04, respectively. Our investigation suggests that the two cationic dyes interact with DNA predominantly via electrostatic interaction.
Immobilization of a cerium redox mediator on a glassy carbon electrode for electroorganic reactions
Authors: Morita Masayuki , Kitamura Shingo , Ishikawa Masashi , Matsuda Yoshiharu
Published: May 2005

Cerium(III) species have been immobilized in Nafion films coated on a glassy carbon electrode by a dipping method. The redox activity was characterized in aqueous acidic solutions by cyclic voltammetry. The redox process was diffusion‐limited, suggesting that the cerium(III) species distributed in the multilayer of Nafion and that the diffusion of the charge‐compensating anion in the film was rate‐controlling. The cerium(III)‐immobilized Nafion films showed a mediation activity for the electrochemical oxidation of substituted benzylalcohols to yield corresponding arylaldehydes and carboxylic acids. The activity and the selectivity of the process depended on the oxidation potential of the substrate compound. Macroelectrolysis of p‐nitrobenzylalcohol using cerium(III)‐immobilized electrodes gave corresponding p‐nitrobenzaldehyde with a high rate and selectivity.
Electrolyte effects on mixed valence state stability of an electropolymerized bis(fulvalene)diiron film
Authors: Nishihara Hiroshi , Nakagawa Toshie , Aramaki Kunitsugu
Published: May 2005

Cyclic voltammetry and spectroelectrochemistry of an electropolymerized film of [Fe2(C10H8)(C10H7CClCHCHO)] (1) were carried out in acetonitrile with Bu4NPF6, Bu4NBF4, Bu4NClO4 or Bu4NHSO4 to determine how the two parameters concerning the stability of the mixed valence state, comproportionation constant, Kc, and mixing coefficient, α, depend on the type of counterions. The difference in electrolyte anions causes slight changes in α exhibiting the magnitude of delocalization of charge between two metal centers. However, significant changes in Kc, suggest that the ion‐pairing and electrostatic repulsion are main factors that alter the electrochemical stability of the mixed valence state.
The determination of ethanol in wine by voltammetry with an internal standard
Authors: Yu Ji Jin , Huang Weiguang , Hibbert D. Brynn
Published: May 2005

A rapid method for the electroanalysis of ethanol is presented that incorporates flow extraction at room temperature, with voltammetric detection and potassium ferrocyanide [K4Fe(CN)6] as internal standard. In 0.1 M NaOH electrolyte, ethanol was oxidised at a platinum comb‐shaped working electrode at −300 mV (vs. a Ag/AgCl reference electrode) and K4Fe(CN)6 was oxidized at +180 mV. The ratio of the anodic peak currents was linear with ethanol concentration in the range of 0.1 to 8.0% (v./v.), and the detection limit (calculated as 3 σ background) was 0.012 % (v./v.) for Osteryoung square wave voltammetry (OSWV) and 0.023 %(v./v.) for cyclic voltammetry (CV). The average extraction efficiency of ethanol from aqueous solutions, at 20 ± 1°C, was 8.5%. The repeatability was in the range of 2.5 to 3.3% RSD (n = 8), and accuracy was in the range of 95.2 to 104.7% for the determination of wine samples. Application to wines compared well with GC and HPLC methods and the nominal ethanol concentration determined by gravimetry. Analytical parameters in CV and OSWV are optimized, and the dependence of the extraction efficiency with temperature and nitrogen gas flow is presented.
Large‐scale fractionation of proteins by isoelectric focusing in a multi‐compartment electrolysis apparatus
Authors: Jonsson Mats , Rilbe Harry
Published: May 2005

A preparative electrolysis apparatus for isoelectric focusing of large amounts of proteins is described. It contains 46 separation compartments, and its total volume is about 7.6 liters. The compartments are closed, and internal cooling and stirring are effected by slow rotation of the whole apparatus in a tank filled with cold water. The apparatus can be run with an electric load of up to 5 kW, and an isoelectric focusing takes 2–3 days. It has been shown that crude pepsin can be purified in this apparatus. Preparations of hemoglobin and bovine whey have also been run in the apparatus. Fourteen grams of whey protein has been completely separated into its main components, serum albumin (pI 4.60), α‐lactalbumin (pI 5.01), and β‐lactoglobulin (pI 5.13–5.23), whereas the latter component was partially separated into its subcomponents A and B.
Photometric evaluation of slab gels I. Data acquisition and image analysis
Authors: Kronberg Harald , Zimmer Hans G. , Neuhoff Volker
Published: May 2005

The photometry of two‐dimensional electrophoretograms is described in some detail. This method has a high sensitivity of 5·10−4 O.D., because it takes advantage of the noise characteristics of photometric signals. Digital image analysis is used to locate and evaluate the individual spots on the electrophoretogram. The presented procedure of segmentation detects the faintest protein spots visible by eye and separates even confluent spots reliably and automatically. The combination of scanning photometry and image analysis is a sensitive, versatile, and accurate method for the evaluation of slab gels.
Fingerprinting of casein digests by isoelectric focusing and SDS electrophoresis
Authors: Righetti Pier Giorgio , Muneroni Paola , Todesco Rosanna , Carini Sandra
Published: May 2005

We have demonstrated the possibility of performing fingerprints in the single isoelectric focusing dimension, able to resolve at least 50 peptide zones, down to a minimum critical length of 12 amino acids. Isoelectric focusing can be coupled to SDS‐gel gradient electrophoresis, provided the fragments have a minimum molecular mass of 3500 daltons. We have applied this system to the analysis of digests of casein micelles. Upon digestion with extracts of lactobacilli, α and β caseins are broken preferentially into two major fragments, a pI 7.1 chain of 10 000 daltons, and a pI 6.4 chain of 14 000 daltons. Upon digestion with an extract of L. jugurti, only two fragments remain undigested, a pI ∼︁ 3 chain of 5500 daltons (probably the 43 to 90 amino acid fragment of α casein) and a pI ∼︁ 9 chain of 4200 daltons (probably the 1 to 36 amino acid sequence of casein).
A device for measuring the segmental voltages along electrofocusing gels
Authors: Jackiw B. Andrew , Chidakel Burt E. , Chrambach Andreas , Brown Ray K.
Published: May 2005

A device for the measurement of segmental voltages along electrofocusing gels made of polyacrylamide has been designed and tested successfully. It allows for continuous monitoring of the changing voltage gradients which occur during electrofocusing. By means of 11 equidistant embedded platinum probes, the voltage drop at any one segment or sequential array of segments can be determined at any time without disturbing the ongoing electrofcusing. The wires are sealed into the wall of a 6 mm diameter (ID) Pyrex gel tube at 1.0 cm intervals. Measurements on a gel containing 2% Ampholine (pH‐range 3.5–10) showed that the voltage gradient changes during electrofocusing. Starting with a uniform voltage drop of 10 ± 2%cm across a 10 cm gel, two transient peaks of high potential (comprising at 15 min 17% and 25% of the applied voltage) move progressively from the gel periphery toward its center. They coalesce into a broad single peak centered at 0.4 of the distance from the anodic gel terminus within 1.5 h. This major single peak remained stationary, changing slightly in magnitude during the next 18 h of electrofocusing. At this final stage, the voltage peak occurred at pH 6.3 and approximately 25% of the applied voltage was across this segment of gel.
A density‐gradient column for rapid preparative isoelectric focusing
Authors: Jonsson Mats , Ståhlberg Jan , Fredriksson Stig
Published: May 2005

In this paper a 112‐ml density‐gradient column is described, which allows a rapid, low‐resolving pre‐focusing over its shorter dimension (20 mm), followed by a high‐resolving fine‐focusing over the longer dimension (112 mm) when the column is turned through 90°. This is possible due to the rectangular cross‐sectional shape of the column and to a special design of the electrode vessels. Test runs made with sperm whale myoglobin have shown that very good separations can be obtained in as little as four hours.
Visualization of proteins with a silver “stain”: A critical analysis
Authors: Poehling Hans‐Michael , Neuhoff Volker
Published: May 2005

Optimal silver staining of proteins in polyacrylamide gels is a fine balance between silver deposition on the proteins and the level of background staining. Reproduciable staining requires empirical standardization of many steps in the procedure. Silver staining in the present form does not stoichiometrically stain proteins, unlike Coomassie Blue. Staining intensity does not bear a linear relationship to protein mass. This is demonstrated by densitometric evaluation of both silver and Coomassie‐stained marker proteins fractioned by sodium dodecly1 sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Although silver staining is more sensitive than Coomassie Blue, the increment in sensitivity is both different for individual proteins and dependent on protein concentration. Nevertheless it is an excellent method for one‐ and two‐dimensional (2‐D) electrophoresis if only minimal amounts of protein are available and only a qualitative or semi‐quantitative evaluation is required.
A sensitive method for determining the nephrotoxic effects of the analgesic acetaminophen upon esterases using isoelectric focusing
Authors: Hennis Hugh L. , Allen Robert C. , Hennigar Gordon R. , Simmons Margaret A.
Published: May 2005

In a study of the biochemical mechanisms of renal toxicity of acetaminophen, quantitative studies of the nonspecific kidney esterases were carried out in New Zealand white female rabbits. After the administration of a sublethal dose of 2.5 g/kg acetaminophen, four animals each plus four controls were sacrificed at 24 and 48 h. subsequent isoelectric focusing of kidney homogenate supernatants over a pH range of 3.5–8.0 showed marked changes in each of the groups. Zymograms of the nonspecific esterases in each group showed a characteristic pattern which deviated greatly from control animals. A morphological and histochemical examination of kidney sections from all animals showed no pathological changes in any of the kidneys. This would indicate that abnormal biochemical changes are being manifested in the kidney long before any morphological deviations. It would seem that this group of enzymes could provide a valuable market for early toxicological changes.
Steady‐state rheoelectrolysis — a method for isoelectric focusing without carrier ampholytes. I. The pH course to be excepted on rheoelectrolysis of a buffer solution composed of a weak acid and its salt with a strong base
Authors: Rilbe Harry
Published: May 2005

Rheoelectrolysis is defined as convection‐free electrolysis of an electrolytic solution under simultaneous liquid transport from anolyte to catholyte and vice versa. Equal rate of transfer in both directions is assumed in this paper, and hence there is no liquid flow within the electrolyser. If this process is conducted long enough, a steady state can be expected, characterised by mutually balancing flows of ion constituents within the electrolyser (by electric migration) and in external ducts (by pumps). The differential equation of the steady state is deduced, and it is shown that it is easily solvable if transport numbers and diffusion coefficients are constant throughout the electrolyser. In this case the concentration courses become linear. The conditions for the presence of both buffer components in all parts of the electrolyser. are given. These conditions being satisfied, it is possible to calculate the pH course to be expected throug the eleltolyer. It is found that a pH span of about 2.6 unite can be covered by the rheoelectolysis of an odinary buffer solution. The pH gradient is at a minimum at the centre of the apparatus and increases towards both electrodes. The pH courses is sigmoid in shape and appears to be very useful for isoelectric focusing of proteins and other ampholytes. The method may become important in preparative work on a large scale in order to save the cost of carrier ampholytes in quantities.
Sex differences in blood protein patterns: Two dimensional electrophoretic analysis of the acidic proteins of rat plasma
Authors: Khirabadi Bijan S. , Gersten Douglas M. , Park Chan M. , Ramey Estelle R. , Ramwell Peter W. , Ledley Robert S.
Published: May 2005

Plasma from normal male and female rats was analyzed, after removal of albumin by affinity chromatography, by two‐dimensional electrophoresis. The results demonstrated gender differences in two plasma proteins with apparent molecular weights of 70 000 and 84 000 and isoelectric points ranging from 6.2 to 6.4, and 5.1 to 5.5, respectively. Both proteins were found to be more concentrated in the plasma minus albumin (P‐alb) fraction of females than males in both Wistar and Sprague‐Dawley rats. The 70 000 protein is serum albumin which was more completely removed from male plasma than female plasma. A possible mechanism responsible for this difference is discussed. The results support the existence of “maleness” and “femaleness” in blood protein patterns of rats.
Application of direct tissue rocket‐line immuno‐electrophoresis to the study of α‐amylase production and its localization in wheat seeds during the early stages of germination
Authors: Daussant Jean , Renard Henii A. , Skakoun Anna
Published: May 2005

Line immunoelectrophoresis was adapted to the quantitative analysis of antigens directly from tissues without any previous extraction. The technique was used in a study on the production and the distribution of two α‐amylase isozymes (I and II) in tissues of wheat seeds that sprouted (radicle emergence) after different periods of germination. The α‐amylase content was examined in the scutellum and in three tissue fractions dissected from a 0.5 mm slice parallel and next to the scutellum (aleurone layer, outer and inner part of starchy endosperm). At the stages of germination investigated, α‐amylase I was not detected in these tissues. The results indicate that α‐amylase II accumulated in the scutellum and also in the aleurone layer after one day's germination; after about two day's germination, the α‐amylase II content in the aleurone layer was similar to or higher than that in the scutellum. Moreover, the production and distribution of α‐amylase II do not seem to be correlated with the radicle emergence.
The agar contact replica technique after isoelectric focusing as a screening method for the detection of enzyme variants
Authors: Pretsch Walter , Charles Daniel J. , Narayanan Komaratchi R.
Published: May 2005

Electrofocusing has been adapted in order to make screening experiments for the detection of enzyme variants more efficient. The principle of the new technique consists in making agar copies of the polyacrylamide gel by contact; the original gel and the agar copies are then developed for different enzyme systems. By doing so, the number of enzymes,i.e.loci, per test animal screened after a single electrofocusing run is increased. This method has proved to be feasible for the detection of mouse variants with altered enzyme banding patterns due to parental treatment with chemical mutagens.
Routine phenotyping of phosphoglucomutase (PGM1) by thin‐layer focusing: Isoelectric points of 14 different variants
Authors: Dykes Dale D. , Copouls Barbara A. , Polesky Herbert F.
Published: May 2005

A routine technique for phenotyping the PGM1 marker system was developed using thin‐layer isoelectric focusing on agarose. Resolution of PGM1 variants by thin‐layer isoelectric focusing on 0.5 mm gels was found to be superior to isoelectric focusing on ⩾ 1 mm gels. Isoelectric points were determined for 14 different PGM1 variants and should serve as a useful tool for comparing variants between laboratories.
Sensitive fluorescence staining for RNA and DNA species separated in polyacrylamide gels
Authors: Neuhoff Volker , Huether Gerald , Engelhardt Uta
Published: May 2005

The fluorescent dye, Hoechst Dye No. 33258, was found to be of excellent quality for staining of not only DNA, as reported in the literature, but also of RNA species after separation in polyacrylamide micro gels. The staining procedure is simple and very sensitive. There are no problems with background staining and under certain conditions the nucleotide‐dye complex is stable for a long time. The fluorescence intensity is linear to the amount of nucleic acid bound by the dye within certain limits.
Electrophoretic separation of human lymphocytes by means of immunomicrospheres
Authors: Smolka Adam , Kempner David , Rembaum Alan
Published: May 2005

Immuno‐labeling of human peripheral blood lymphocytes with either polyglutaraldehyde or polyacrolein microspheres was shown by analytical electrophoresis to generate two electrokinetically distinct sub‐populations of lymphocytes. By means of free‐flow electrophoresis, the high mobility fraction was found to consist predominantly of T lymphocytes using an anti‐T cell antibody, while the low mobility fraction consisted of lymphocytes labeled with immunomicrospheres, identifying these cells as B lymphocytes. The immunomicrosphere‐induced reduction of B‐cell mobility below that of the T cells thus allowed the enrichment and recovery of viable T cells in preparative quantities by free‐flow electrophoresis.
Detection of specific bovine serum albumin‐sodium n‐dodecyl sulphate complexes by polyacrylamide gel electrophoresis
Authors: Dominguez‐Reboiras M. , Jones M. N.
Published: May 2005

Polyacrylamide gel electrophoresis has been adapted to investigate bovine serum albumin (BSA) at sub‐saturation concentration levels of sodium n‐dodecyl sulphate (SDS). Two specific complexes were detected which coexisted between SDS concentrations of 0.4 and 5 mM. The binding isotherm for SDS on BSA was determined under comparable conditions by equilibrium dialysis and used in conjunction with the electrophoretic data to determine the composition of the complexes. The complexes had the following stoichiometry, BSA (SDS)5 and BSA (SDS)36, and closely resembled two of the three complexes that have been observed by moving boundary electrophoresis. The experiments indicate that the polyacrylamide gel electrophoresis (PAGE) technique when applied below surfactant saturation could be a useful tool in the study of protein‐surfactant interactions.
Computerized analysis of double‐label autoradiographs: A screening method for 1,25‐dihydroxyvitamin D3 induced changes in intestinal protein synthesis
Authors: Kendrick Nancy C. , Eakle Kurt , Deluca Hector F.
Published: May 2005

The McIDAS computer system (Man‐computer Interactive Data Access System) at the University of Wisconsin was used to analyze double‐label autoradiographs of two‐dimensional (2‐D) electrophoresis gels. The 4 samples used to generate the 2‐D gels were obtained from 8 vitamin D‐deficient rat intestines 25 h after injection with 1,25‐dhydroxyvitamin D3 (1,25‐(OH)2 D3) or vehicle. To perform the analysis, the patterns on Kodak XAR5 (XR) film, which detects 14C + 3H by fluorography at ‐75 °C, and on Kodak NS‐2T (NS) film, which detects 14C only by direct autoradiography at room temperature, were converted to digital images. The McIDAS system was used to multiply the NS image by a normalization factor which matched it to the XR image with respect to exposure. After background subtraction from both images, the XR image was divided by the aligned normalized NS image; the resultant ratio image was multiplied by an enhancement factor and colored according to pixel brightness. Polypeptides with a high or low ratio relative to the average ratio stood out clearly in contrasting colors on the ratio image and could be detected by visual inspection. The actual XR/NS density ratios of these highlighted spots could then be quantitated by spot integration. To test the method, XR/NS density ratios were measured for 34 spots on each of the 4 films, sets and the spot ratios compared to the spot appearances on the ratio image. With the exception of a few false positives, agreement was good. Several polypeptides were found to be increased by 25–50 % relative to the average ratio while one polypeptide of molecular weight 50 000, approximate pI 5.5, was increased an average of 240 %.
Formation of natural pH gradients in sequential moving boundary systems with solvent counterions. I. Theory
Authors: Hjelmeland L. M. , Chrambach A.
Published: May 2005

The moving boundary theory has been applied to the special case in which the proton and hydroxyl ion are the sole counterions. Moving boundaries between two monovalent weak acids, two biprotic acidic ampholytes, two weak bases and two biprotic basic ampholytes were considered. This application of the theory leads to constituent concentration relationships, values for pH and conductance of each phase and step pH gradients when sequential moving boundaries are considered. In addition, rigorous conditions for achieving the steady‐state in such systems are also presented. Application of the theory allows for the prediction of the formation and displacement of pH gradients. In contrast to the classical theory of pH gradients formed by ampholytes, the establishment of inverse pH gradients and the electrophoretic displacement of natural pH gradients is clearly accounted for.
The distribution of and the biochemical and serological relationships between the I/i and ABH blood group antigens of the human erythrocyte membrane as determined by immunoelectrophoretic techniques
Authors: Oppenheim Joel D. , Nachbar Martin S. , Blank Margild
Published: May 2005

Macromolecules isolated from human erythrocyte membranes which expressed I/i and ABH blood group activities were analyzed by quantitative immunoelectrophoretic techniques in order to determine the distribution of and the relationship between these two blood group systems. To help in these studies a rocket immunoelectrophoretic assay, with a maximum sensitivity of 10 ng, was developed which could detect and quantitate erythrocyte macromolecules which expressed I/i and A and B blood group determinants. In addition, a modification of the tandem crossed immunoelectrophoresis technique was also used to elucidate the structural relationship between determinants of these systems on one of the isolated macromolecular species. Serological and other immunological data are presented which throughly support the immunoelectrophoretic results. From these studies we have concluded that I and most likely i antigenic determinants are primarily expressed on poly(glycosyl)ceramides (i. e. macroglycolipids) and possibly to a lesser extent on Band 3. I active poly(glycosyl)ceramide could be isolated in equal quantity from any adult ABH type erythrocyte; i activity was found in similar quantity in the same fraction isolated from one i adult erythrocyte population. Besides expressing I/i activity the various poly(glycosyl)ceramide fractions also exhibited A, B or H activity as well, dependent upon the ABH cell type from which they were isolated. Unlike the equal distribution of I, however, ABH activity was type‐specific in that the poly(glycosyl)ceramide isolated from type A (either A or Ai), B or 0 cells could react only with anti‐A, anti‐B, or Ulex anti‐H lectin, respectively. Using the modified tandem crossed immunoelectrophoresis (CIE) technique, it was established that the same glycolipid molecules which carry I blood group activity express the A and B determinants as well. Band 3 material isolated from 0 erythrocytes exhibited I, i and H activities. From CIE analysis the I activity of Band 3 appeared to reside in a subpopulation of this material.
Detection of the major non‐serum derived proteins of body fluids by immunodeletion and two‐dimensional gel electrophoresis
Authors: Dermer Gerald B. , Edwards Jesse J.
Published: May 2005

Commercial antisera to human serum were coupled to Sepharose 4B or Staphylococcus aureus bacteria and used as immunoadsorbents to separate the serum‐derived proteins in several types of body fluids from the fluid‐specific, non‐serum derived proteins. Two‐dimensional gel analysis of fluids after incubation with one or the other immunoadsorbent produced similar results. Cerebrospinal, prostatic and amniotic fluid each exhibited a characteristic array of probable fluid‐specific protein families which were not detected in serum. Data from model experiments suggested that Sepharose 4B was a somewhat better matrix material than Staphylococcus aureus cells. Pretreatment of body fluids with anti‐human serum immunoadsorbents should facilitate the detection of fluid‐specific protein markers of disease in two‐dimensional gels.
Agarose gel electrophoresis of bacteriophages and related particles. III. Dependence of gel sieving on the agarose preparation
Authors: Serwer Philip , Allen Jerry L. , Hayes Shirley J.
Published: May 2005

The sieving of different preparations of agarose has been measured during agarose gel electrophoresis, using as samples a spherical bacteriophage and two spherical bacteriophage‐related particles with radii between 13.3 and 41.9 nm. Semilogarithmic plots of electrophoretic mobility as a function of agarose perentage (A) were linear for A ⩽ 0.85, but became progressively nonlinear as A increased above 0.85. The regions of linearity in these plots were used to measure gel sieving and to determine an effective gel fibre radius. The sieving of the different preparations increased in the order HGT[P] < ME < Seaplaque (hydroxyethylated) Isogel (additive containing) < SeaPrep 15/45 (hydroxyethylated) (trade names of preparations from Marine Colloids). However, the effective gel fibre radius did not differ significantly from 25 nm among HGT[P], ME, SeaPlaque and SeaPrep 15/45 agarose; for Isogel, this radius was 20 nm. These data have been interpreted to indicate that the comparatively high sieving of SeaPlaque, SeaPrep 15/45, and to some extent Isogel agarose was caused by a comparatively low agarose mass per fibre volume and high total fibre length per gel volume. For at least some particles smaller than those used here, it is likely that migration through the gel fibres detected here occurs during agarose gel electrophoresis.
Isoelectric focusing in immobilized pH gradients in presence of urea and neutral detergents
Authors: Gianazza Elisabetta , Artoni Gianfranco , Righetti Pier Giorgio
Published: May 2005

The pK's of Immobilines, the reactive acids and bases used for establishing the pH in polyacrylamide gel matrices for isoelectric focusing with immoblized pH gradients, were measured in presence of urea (up to 8 M) and a neutral detergent'(2 % Nonidet P‐40). The temperature dependence of the pK's in 8 M urea was also investigated. ΔpK's due to the addition of urea were found to be higher for the acidic than for the basic Immobilines. Formulations are given to obtain 4 and 5 pH unit wide gradients with pre‐fixed extremes and linear in the presence of 8M urea. Such gels require, as pretreatment before the run is started, either an electrophoretic elution, by applying the voltage perpendicular to the direction of the pH gradient, or extensive washing in water, followed by drying and re‐equilibration in urea solution.
Porous support for electroblot transfers
Authors: Saravis Calvin A. , Cook Richard B. , Polvino William J. , Sampson Christian E.
Published: May 2005

Porous supports for electroblot transfers of human tumor associated antigens from agarose slab gels allow easier slab gel handling and minimize possible distortion of the protein transfers.
Polymorphism of soluble rat brain guanylate cyclase is produced during isoelectric focusing
Authors: Basset Paul , Zwiller Jean , Revel Marie‐Odile , Vincendon Guy
Published: May 2005

Soluble guanylate cyclase purified from rat brain exhibits about 30 different components between pH 5.3 and 6.5 on polyacrylamide gel isoelectric focusing (IEF) Specific immunodetection after IEF of guanylate cyclase from fresh rat brain supernatant allows the detection of a complex profile, indicating that the complex pattern observed with purified enzyme is not related to sample aging. In contrast, purified enzyme gives only one major band on electrophoresis in presence or absence of sodium dodecyl sulfate and a single line of immunoprecipitation was obtained on crossed immunoelectrofocusing. The IEF pattern exhibited by guanylate cyclase is dependent on the sample concentration, the pH range of sample application, the carrier ampholytes used and on the presence of urea in the gel. Fractionation tests, performed with partially purified enzyme or with purified enzyme, indicate that this complex pattern is produced during IEF and does not correspond to an inherent heterogeneity of soluble rat brain guanylate cyclase. The distribution of carrier ampholytes used to form the pH gradient and that of guanylate cyclase bands was found to be very similar at the steady‐state of IEF, suggesting that interaction between carrier ampholytes and rat brain soluble guanylate cyclase can be involved in the generation of the complex IEF pattern.
Artefacts associated with Nonidet P‐40 upon sodium dodecyl sulphate polyacrylamide gel electrophoresis of protein
Authors: Marshall Thomas
Published: May 2005

One‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of protein in the presence of Nonidet P–40 (NP–40) induces vertical streaking which breaks horizontal bands of protein to produce a “multiple spot” effect. The same effect can be induced upon high resolution two‐dimensional electrophoresis if the NP–40 concentration of the first‐dimension isoelectric focusing (IEF) gels is increased to 4 % w/v and the gels electrophoresed (SDS‐PAGE) without SDS‐equilibration. The relevance of this phenomenon as regards interpretation of two‐dimensional electrophoretic patterns is discussed.
Electrophoretic demonstration of interactions between group specific component (vitamin D binding protein), actin, and 25‐hydroxycholecalciferol
Authors: Emerson David L. , Galbraith Robert M. , Arnaud Philippe
Published: May 2005

Group specific component (Gc) is known to interact with both vitamin D3 metabolites and actin. The present study was undertaken to determine if analytical isoelectric focusing could form the basis of a simple and reliable method for discriminating native Gc and complexes with 25‐hydroxycholecalciferol (25(OH)D3) from those formed with actin and with both ligands. Increasing amounts of G‐actin and/or 25(OH)D3 were added to purified Gc of both Gc1 and Gc2 phenotypes. Actin and 25(OH)D3 interacted independently and simultaneously with native Gc, giving rise to three different complexes of increasing acidity: Gc‐25(OH)D3, Gc‐actin, and actin‐Gc‐25(OH)D3, which were clearly resolved from the native Gc protein and from each other. In addition, the inherent differences in microheterogeneity and polymorphism between the two major phenotypes examined (Gc1 and Gc2) were also retained in their respective complexes. Similar results were obtained upon addition of 25(OH)D3 or actin to whole human serum, the bands corresponding to native or complexed Gc being recognized in this case by print immunofixation. These results indicate that complexes formed between Gc protein and both 25(OH)D3 and actin can be clearly detected and resolved by electrophoretic procedures.
Analysis of normal human aqueous humour proteins by two‐dimensional gel electrophoresis
Authors: Segers Jeanine , Rabaey Marcel , Van Oye Rafaël
Published: May 2005

High resolution two‐dimensional electrophoresis combined with a sensitive silver stain has been used to study the proteins of normal human aqueous humour. The protein patterns of small, unconcentrated, individual samples and of pooled and concentrated aqueous humour samples were compared with plasma or serum from the same individual or with a normal serum. The protein maps of aqueous humour and serum showed great similarity. More than 30 groups of spots were visualized in aqueous humour after silver staining and 18 of them have been tentatively identified. Three polypeptide groups were observed which were not found in plasma or serum. The different clusters, which appeared in the region of high (> 90 000), medium (50 000–45 000) and low (25 000–15 000) molecular weight polypeptides, remain unidentified. The most abundant cluster represents a molecular weight of approximately 48 000.
Computer‐aided standardization of two‐dimensional electrophoresis: Improvement of biophysical description of proteins in relative versus absolute coordinate systems
Authors: Fosslien Egil , Prasad Rameshwar , Stastny Jaroslav
Published: May 2005

Protein migration distances on two‐dimensional (2‐D) gels were measured in mm (absolute) and in relative internal standard units using creatine kinase carbamylation trains (x‐axis) as relative charge (CK‐Cr) scale and rat heart extract lines as relative molecular weight (Mr) scale (y‐axis). Variation in absolute protein location generally increases with increasing migration distance in both axes with 6–12 mm and 5–11 mm for selected spots in the x‐ and y‐axis respectively; variation in relative migration is significantly less, ranging from 0.9–2.5 mm and 3–4 mm in the x‐ and y‐axis respectively; data are from seven separations run in the same batch, measurements obtained from 1.6 × magnified projections of photographed gels. A computerized plotter was used to generate scaled relative charge ‐ relative molecular weight (Cr‐Mr) plots for each separation based upon absolute positions of internal protein standard spots (first dimension) and lines (second dimension). The results support the use of computer‐delineated‐relative coordinate systems (Cr‐Mr plots) for biophysical description of proteins separated by 2‐D electrophoresis.