bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential flow‐injection potentiometric determination of iodide and iodine in povidone iodine pharmaceuticals
Authors: Hassan Saad S. M. , Marzouk Sayed A. M.
Published: May 2005

A potentiometric sensor‐based flow‐injection system for determining iodide is described. The system utilizes a flow‐through sandwich cell and incorporates iron (II)‐tris bathophenanthroline iodide ion pair complex as an exchange site in a PVC matrix membrane. The sensor displays a fast response for 10−1 to 10−6 M iodide ion with an anionic slope of 59.1 ± 0.3 mV/concentration decade over the pH range 1 to 11. The experimental setup offers a precise, sensitive, selective and high sample throughput technique for determining iodide and iodine over a wide range of concentration. The limit of detection is 0.5 ppm iodide in a 20 μL sample. The average recovery is 99.8%, and the mean standard deviation is 0.5%. Sequential flow‐injection determination of iodide and iodine in some pharmaceutical preparations containing povidone iodine is performed by sample injection in a sodium sulfate‐ascorbic acid stream and monitoring of the total iodide equivalent to I− and I2. This is followed by injection of a second sample in an alkaline resorcinol stream and measuring of the iodide equivalent to I− and 1/2 I2. Iodine and iodide concentrations are calculated from the consecutive signals. The method gives results in good agreement with those obtained by the United States Pharmacopeia.
Electrochemical determination of iron in YBa2(Cu1–xFex)3O7 superconducting compounds
Authors: Boutakhrit Khalid , Kasmi Asma El , Kauffmann Jean‐Michel , Deltour Robert , Mehbod Mohamed , Marvin Craig A.
Published: May 2005

The electrochemical determination of iron in doped YBaCuO superconducting compounds has been investigated by differential pulse polarography (SMDE) and by differential pulse voltammetry using a glassy carbon electrode. A well‐defined peak for iron was observed at –0.15 V (vs. Ag‐AgCl) reference electrode in sodium citrate/EDTANa2H2 pH 6. The electrochemical results have been validated by spectroscopic procedures, including atomic absorption and energy dispersion X‐ray spectroscopy. The differential pulse polarography using the standard addition method for peak validation has been found to be most appropriate.
Comparison of dibenzo‐16‐crown‐5 compounds with pendent amide groups as sodium ionophores in ion‐selective electrodes and in solvent extraction
Authors: Ohki Akira , Iwaki Kaoru , Naka Kensuke , Maeda Shigeru , Collier James J. , Jang Youngchan , Hwang Hong‐Sik , Bartsch Richard A.
Published: May 2005

Potentiometric selectivities for alkali metal, alkaline earth metal and ammonium ions for a series of N,N‐dialkylsym‐( R)dibenzo‐ 16‐crown‐5‐oxyacetamides in solvent polymeric membrane electrodes are determined. For N, N‐dipentyl sym‐(R)dibenzo‐ 16‐crown‐5‐oxyacetdamides, the Na+/K+selectivity increases with variation of the R group in the order: hydrogen « propyl (linear alkyl) < isopropyl, neopentyl (branched alkyl). Solvent extractions of sodium and potassium picrates from aqueous solutions into 1,2‐dichloroethane were also performed. Ratios of the extraction constants for Na+and K+(logK/K) are similar in magnitude and show the same trends for structural variation within the ionophore as do the potentiometric selectivities between Na+and K+(logK) in the polymeric membrane electrodes. From the picrate extraction results. it is deduced that the enhancement in potentiometric selectivity for Na+over K+when the R group is changed from hydrogen to propyl arises from increased binding of Na+and diminished complexation of K+. On the other hand, the enhanced potentiometric selectivity for Na+over K+when a linear alkyl R group is replaced by a branched alkyl group appears to result from decreased complexation of K+only.
Stand for high pressure electrochemical measurements
Authors: Górecki J. , Gołaś J.
Published: May 2005

The complete stand for a high pressure electrochemical experiment (up to 0.8 GPa), construction of the electrochemical cells and the high pressure chamber is presented. The procedure of a high pressure voltammetric experiment is described with some practical advice on how to prepare and maintain the electrodes.
Electrochemical transduction of interactions of atrazine with bilayer lipid membranes
Authors: Nikolelis Dimitrios P. , Andreou Vangelis G.
Published: May 2005

This work investigates the interactions of atrazine with bilayer lipid membranes (BLMs) that can be used for the direct electrochemical sensing of this herbicide. Egg phosphatidylcholine (PC) and dipalmitoylphosphatidic acid (DPPA) were used for the formation of solventless BLMs. The interactions of atrazine with these membranes were found to be electrochemically transduced by BLMs in the form of a transient current signal with a duration of seconds, which reproducibly appeared within 1 min after exposure of the membranes to atrazine. The sensitivity of the response was maximized by use of BLMs composed of 35% (w./w.) DPPA, and by alteration of the phase distribution within membranes by the introduction of calcium ions in bulk solution. The mechanism of signal generation was related to the adsorption of atrazine with a consequent rapid reorganization of the membrane electrostatics due to atrazine aggregation at the surface of BLMs. Hydrogen bonding between atrazine and the carbonyl group of the lipid was explored by addition of platelet‐activating factor (PAF; an ether analog of PC) in BLMs composed of PC. Differential scanning calorimetry of vesicles composed of 15% DPPA was used to study the aggregation of atrazine in membrane domains enriched in the charged lipid. The magnitude of the transient current signal was linearly related to the concentration of atrazine in bulk solution with sub‐micromolar detection limits. This electrochemical transduction of atrazine interactions with BLMs holds prospects for flow injection monitoring of triazine herbicides.
Electrogeneration of superoxide ion at mercury electrodes with a hydrophobic adsorption film in aqueous media
Authors: Matsumoto Futoshi , Tokuda Koichi , Ohsaka Takeo
Published: May 2005

The electrogeneration of superoxide ion (O) by the one‐electron reduction of O2in alkaline aqueous media has been examined using the hanging mercury drop electrode in the presence of several surfactants of the quinoline groups. Based on the superoxide dismutase (SOD)‐catalyzed disproportionation behavior of Oon the cyclic voltammograms, new surfactants, in the presence of which Owas actually electrogenerated, could be searched: 2‐, 4‐, 6‐, 7‐ and 8‐methylquinolines (3,4,5,6 and 7), and 2‐phenyl and 5,6‐benzoquinolines (8 and 9). It was found that the effectiveness for the electrogeneration of Olargely depends on the surfactant and decreases in the order of quinoline (l), isoquinoline (2) > 5, 6, 7 > 3, 4 > 8 > triphenylphosphine oxide (10) > 9. In addition, a brief discussion on the structure and physicochemical properties of the surfactant film desirable for the effective Oelectrogeneration in aqueous media is given.
The effect of the removal of trace impurities on the adsorption behavior of octanoic acid at mercury–solution and at air–solution interfaces
Authors: Avranas Antonis , Retter Utz , Lunkenheimer Klaus , Lohse Heinz
Published: May 2005

In this article we explore the kinetics of adsorption of octanoic acid at the mercury‐water interface using phase sensitive AC voltammetry. These measurements were performed before and after stepwise purification of the octanoic acid solution, in order to distinguish between the adsorption behavior of the octanoic acid and its impurities. The adsorption of both octanoic acid and the surface active impurities decrease the capacitance with time. Removing the latter during the purification procedure results in a faster achievement of the equilibrium state and in higher capacitance values. The adsorption was also studied at the air‐water interface using dynamic surface tension measurements. Comparable results are obtained for both interfaces.
Bioelectrocatalytical studies of the effect of some pharmaceuticals on the acetylcholinesterase activity
Authors: Stoytcheva Margarita , Zlatev Roumen
Published: May 2005

A bioelectrocatalytical method is applied to study the influence of some pharmaceuticals (atropine, caffeine, thiamine, nicotinic acid, quinine and eserine) on the acetylcholinesterase activity. The measuring principle is based on the process of acetylthiocholine hydrolysis, catalyzed by the acetylcholinesterase, immobilized on a graphite electrode surface. The product of this reaction is electroactive and its oxidation current is registered. The kinetics of the process, occurring with the participation of the above mentioned substances, is studied. It is found that competitive (eserine, nicotinic acid, atropine, caffeine) and noncompetitive (thiamine, quinine) inhibition takes place; kinetic parameters of the enzymic reaction (apparent Michaelis constants and constants of inhibition) are determined using electrochemical data, in the absence and in the presence of the inhibitors. It is observed that some of the tested substances behave as activators, under certain conditions. It was found that atropine, caffeine, thiamine, nicotinic acid and quinine do not show any electrochemical activity under the described conditions. Only eserine is oxidized; this however, does not interfere with the enzyme determination in a certain concentration range. Calibration plots, useful for the quantitative analysis, are constructed.
Anodic differential pulse voltammetric analysis of iodide with a cinchonine‐modified carbon paste electrode
Authors: Kwak Myung‐Keun , Park Deog‐Su , Won Mi‐Sook , Shim Yoon‐Bo
Published: May 2005

The stripping voltammetric determination of iodide ions has been completed using a cinchonine modified carbon paste electrode. Iodide ions were deposited onto a carbon paste electrode containing cinchonine via the ion pairing reaction in an acidic medium. The oxidation peak potential of the iodide ion is + 0.65 V. The analytical conditions for the determination of the iodide ion were optimized by cyclic voltammetry. The optimal composition of CMCPE and pH of the solution on the deposition of iodide are 40%(w/w) and 2.15, respectively. Differential pulse voltammetry was applied to determine the calibration plot and detection limit. The detection limit was 8.0 × 10−8M for 20 min of deposition time and relative standard deviation (5 determinations) was ±4.2% at a 7.0 × 10−7M iodide solution. The interferences of anions and cations were investigated. The thiosulfate ion interferes with the oxidation current of the iodide ion by about 12%. C1−, Br−, SCN−, C2O42−, CIO4−, NO3Co2+Fe(CN)63−, Ni2+, Cu2+, Cd2+, Pb2+, and Hg2+do not interfere with the determination for the iodide ion. The applicability of the CMCPE to the determination of iodide in a disinfectant has also been investigated.
Carbon electrodes in amine analysis: Effect of chemically modified carbon surfaces on signal quality and reproducibility
Authors: Surmann Peter , Peter Bernhard
Published: May 2005

The preparation of chemically modified graphite electrodes by various chemical treatments is described. These electrodes were tested with chlorpromazine, cyclopiroxolamine, sulfathiazole, bamipine, sparteine and diphenhydramine by differential pulse voltammetry. Compared with the unmodified and freshly polished carbon electrodes, the modified ones exhibit attractive properties such as enhanced stability against film formation. The initial signals at untreated electrodes are very high and decrease rapidly, but at pretreated electrodes the stability of the signals is usually improved. Adsorption and poisoning by film formation is suppressed by giving the surface of the electrode a chemical identity by chemical pretreatment. The improvements are not general but individual ones.
Electroanalytical sensors for nonconducting media based on electrodes supported on perfluorinated ion‐exchange membranes
Authors: Bontempelli Gino , Comisso Nicola , Toniolo Rosanna , Schiavon Gilberto
Published: May 2005

Electroanalytical sensors, suitable for the analysis and monitoring of electroactive analytes present in gaseous phase or low‐conductive liquid media, and based on electrodes in close contact with perfluorinated ion‐exchange polymers are reviewed. The basic operative mechanism of these sensors, in which ion‐exchange polymers act as solid polymer electrolytes (SPE's), is thoroughly discussed, while stressing the fundamental reasons why their behavior differs from that of conventional membrane electrodes. The procedures for preparing composite working electrodes by coating one side of ion‐exchange membranes with stable porous films of conductive materials are described, along with the most common strategies followed to assemble this type of sensors. Useful examples of measurements in electrolyte‐free media of inorganic and organic electroactive species of interest mainly for environmental analysis are given. Future prospects for the development of these sensors are also discussed.
Analytical applications of poly(3‐methylthiophene)‐coated cylindrical carbon fiber microelectrodes
Authors: Agüí L. , Yáñez‐Sedeño P. , Pingarrón J. M.
Published: May 2005

Some analytical applications of carbon fiber microelectrodes (CFMEs) modified with cyclic voltammetric electrogenerated poly(3‐methylthiophene) (P3MT) coatings are described. Cyclic voltammograms in aqueous solutions of the phenolic antioxidants tert‐butylhydroxyanisole (BHA), tert‐butylhydroquinone (TBHQ), propylgallate (PG), and tert‐butylhydroxytoluene (BHT) showed that electrocatalytic effects and an improved electrode kinetics occurred at the modified microelectrodes when compared with GC electrodes of conventional size. Moreover, reproducible voltammograms were obtained, obviating the need of cleaning or pretreatment of the coated microelectrode. Another important analytical advantage was the greatly improved resolution for mixtures of analytes. The behavior of P3MT‐CFMEs in organized media was tested by considering their voltammetric response towards the electrochemical oxidation of PG. The nonionic surfactant Pluronic F 68 was selected as the most suitable to form micellar solutions of PG, as well as the emulsifying agent for the preparation of oil‐in‐water emulsions. A 20:80 ethyl acetate:n‐hexane mixture was used as the organic phase and a 0.05 mol L−1 H3PO4/H2PO4− buffer solution of pH 2.0 as the aqueous continuous phase. Cyclic voltammetry demonstrated that the PG oxidation current was purely diffusion‐controlled in the organized medium. The analytical characteristics obtained by using differential pulse voltammetry were slightly better than those obtained in aqueous solution. Furthermore, the improved resolution for mixtures of analytes is retained in the oil‐in‐water emulsified medium. The proposed method was applied with good results to the determination of PG in spiked commercial dehydrated soup samples by direct emulsification of aliquots of the sample extract in the ethyl acetate:n‐hexane mixture.
A PVC‐based pentathia‐15‐crown‐5 membrane potentiometric sensor for mercury(II)
Authors: Gupta Vinod K. , Jain Suresh , Khurana Upendra
Published: May 2005

Poly(vinyl chloride) (PVC)‐based membrane of pentathia‐15‐crown‐5 exhibits good potentiometric response for Hg2+ over a wide concentration range (2.51 × 10−5 to 1.00 × 10−1 mol dm−3) with a slope of 32.1 mV per decade of Hg2+ concentration. The response time of the sensor is as fast as 20 s. The electrode has been used for a period of six weeks and exhibits fairly good discriminating ability towards Hg2+ in comparison to alkali, alkaline and some heavy metal ions. The electrode can be used in the pH range from 2.7 to 5.0.
Continuous flow and flow injection stripping voltammetric determination of silver(I), mercury(II), and bismuth(III) at a bulk modified graphite tube electrode
Authors: Ye Ruidong , Khoo Soo Beng
Published: May 2005

The epoxy‐impregnated graphite tube electrode bulk modified with 2‐mercaptobenzoxazole, employed in a wall‐jet configuration, was found to be useful for the continuous flow and flow injection stripping voltammetric determinations of AgI, HgII and BiIII. For continuous flow, detection limits for AgI, HgII and BiIII were 1.8 × 10−10 M, 1.9 × 10−9 M and 9.5 × 10−9 M, respectively (10 min accumulation, S/N = 3). Precisions for 5.00 × 10−9 M AgI, 1.00 × 10−8 M HgII and 1.00 × 10−7 M BiIII were 10.5%, 5.77 % and 7.90% (relative standard deviations, n = 6), respectively. In the case of flow injection stripping, with a 500 μL injection loop, detection limits of 0.59 ng, 2.0 ng and 120 ng were obtained for AgI, HgII and BiIII, respectively (S/N = 3). Selected metal ions, inorganic and organic substances were investigated for interferences. The electrode was tested with a certified sample and then applied to the determinations of the metal ions in a urine and a sea‐water sample.
Nitrate ion‐selective sensor based on electrochemically prepared conducting polypyrrole films
Authors: Sun Bianting , Fitch Peter G.
Published: May 2005

Nitrate‐doped polypyrrole (PPy) films on a glassy carbon substrate have been prepared electrochemically in aqueous, acetonitrile, and propylene carbonate solutions for use as nitrate sensors. Lithium nitrate, sodium nitrate, nitric acid, tetraethylammonium p‐toluene sulfonate (TS), and tetradodecylammonium nitrate (TDN) were employed as electrolytes. The effect of dibutylphthalate (DBP) as a plasticizer on the sensitivity and lifetime of PPy film sensors was also investigated. A Nernstian behavior with a slope of 56.9 m V/decade over 0.1–7.4 × 10−5 M NO and a detection limit of 4.7 × 10−5 M were observed for the polymer sensor prepared in acetonitrile solution containing lithium nitrate and 15% plasticizer (DBP). A lifetime of more than 6 months for this PPy film electrode was obtained.
Catalytic polarographic prewave of cobalt(II) induced by carbendazim. Application to the voltammetric determination of benomyl
Authors: Alvarez Jesús L. Muñíz , Calzón Josefa A. García , Fonseca Juan M. López
Published: May 2005

Methyl 1H‐benzimidazol‐2‐ylcarbamate (carbendazim or MBC) catalyzes the reduction of CoII at mercury electrodes in sodium barbital/nitric acid/sodium chloride media. On the basis of a detailed study of the CoII prewave induced by carbendazim, a mechanism for the electrode reaction is proposed and adequate conditions for the analytical determination of carbendazim by differential pulse voltammetry are established. Based on this finding, a voltammetric method for the indirect determination of methyl[1‐(butylcarbamoyl)‐1H‐benzimidazol‐2yl]carbamate (benomyl) is described, following the conversion of benomyl to carbendazim.
Calibration of denaturing agarose gels for molecular weight estimation of DNA: Size determination of the single‐stranded genomes of parvoviruses
Authors: Snyder Cathrine E. , Schmoyer Richard L. , Bates Robert C. , Mitra Sankar
Published: May 2005

Vertical slab gel electrophoresis of DNA with CH3HgOH‐containing agarose produces sharp bands whose mobilities are suitable for size estimation of single‐stranded DNA containing 600–20 000 bases. The relationship of electrophoretic mobility to size of DNA over this range is a smooth, S‐shaped function, and an empirical model was developed to express the relationship. The model involves terms in squared and reciprocal mobilities, and produced excellent fit of known standard markers to measured mobilities. It was used to estimate the sizes of six parvovirus DNAs: Kilham rat virus (KRV), H‐1, LuIII, and minute virus of mice (MVM) DNAs had molecular weights of 1.66 – 1.70 × 106, while the molecular weight of bovine parvovirus (BPV) DNA was 1.84 × 106 and that of adenoassociated virus (AAV) DNA was 1.52 × 106.
Tritium/PPO gel fluorographic efficiency is reduced by Coomassie Blue staining
Authors: Higgins Ratchford C. , Dahmus Michael E.
Published: May 2005

When [3H]‐labeled proteins are analyzed by polyacrylamide gel electrophoresis, the sensitivity of fluorograhic detection following gel impregnation with PPO (2,5‐diphenyloxazole) in DMSO (dimethylsulfoxide) is significantly reduced if the gel has been stained with Coomassie Brilliant Blue R‐250. Since the reduction in fluorographic efficiency depends on the concentration of stain, which in turn depends upon protein concentration, substantial errors may enter into quantitative measurements of fluorograms depending on sample composition. Another reduction in fluorographic efficiency results from reduced penetrability of the gels to PPO when a build‐up of methanol in DMSO and DMSO/PPO solutions is allowed. This loss of fluorographic sensitivity should be sample independent, however.
High resolution preparative isoelectric focusing in layers of granulated gels
Authors: Frey Manuela D. , Radola Bertold J.
Published: May 2005

Discontinuous isoelectric focusing in layers of granulated gels is used to separate up to 1000 mg protein with a resolution of 0.01‐0.015 pI. High resolution is achieved with (i) gel layers of reduced thickness (1 mm), (ii) high field strength in the final stage of focusing (100‐200 V/cm), (iii) long separation distances, and (iv) high Volthour (Vh) products ensuring steady state conditions. We describe approaches to rapid focusing based on either short separation distances or extensive prefocusing with resultant short residence times of the separated substances in the gel. The optimum load capacity is 3‐5 mg protein/ml gel‐bed volume while 10 mg protein/ml gle‐bed volume are still well tolerated. At high loading, major components are seen in the gel as transulcent zones which can be easily identified and harvested. Best results are obtained with Sephadex G‐200 (Superfine) or Bio‐Gel P‐60 (minus 400 mesh). Protein visualization with a new printing technique empolying cellulose acetate membranes requires only 2‐3 min and is insensitive to protein overloading and adhering gel particles. A simple and efficient method is described for the electrophoretic removal of carrier ampholytes from the focused proteins. High resolution preparative isoelectric focusing should prove attractive for many applications due to its great flexibility with respect to sample size, gel volume, separation time, protein recovery, ease of operation and reasonable price.
Isolation and partial characterization of rat α1‐antitrypsin
Authors: Thisner Danuta , Rosengren Åke
Published: May 2005

α1‐Antitrypsin was isolated from rat serum by salting out with ammonium sulfate in three steps, followed by repeated ion exchange on DEAE‐Sephadex A 50 and affinity chromatography on Affi‐Gel Blue. The fractionation was paced with isoelectric focusing in polyacrylamide gels with pH range 3.5‐9.5. The resultant protein was homogeneous in crossed immunoelectrophoresis using rabbit antiserum against whole rat serum. Isoelectric focusing in α1‐antitrypsin phenotyping plates with pH range 4–5 showed a clear five‐band pattern of isolated protein (isoelectric points 4.41, 4.48, 4.50, 4.58 and 4.61, respectively). Immunofixation and zymogram techniques were used to identify the α1‐antitrypsin bands. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis two bands appeared. They had relative mobilities corresponding to apparent molecular weights of 50 000 and 56 000.
Evaluation of the capacity of gel electrophoresis, steadystate and transient state electrofocusing to resolve native human des‐angiotensin‐I‐angiotensinogen from angiotensinogen
Authors: Auzan Colette , Genain Claude , Corvol Pierre , Menard Joël , Chrambach Andreas
Published: May 2005

The resolution of angiotensinogen (Mr56 800) linked to a decapeptide, angiotensin I (Mr 1295), from the same protein without the decapeptide presents a challenging separation problem. Polyacrylamide gel electrophoresis at an optimized pH of 6.88 is unable to distinguish between the two proteins with statistical significance on the basis of size, but is able to discriminate between them with significance on the basis of their net charge difference. Correspondingly, the proteins are separated in gel electrofocusing with a pI difference of about 0.1 pH unit. For practical purposes, neither the charge separation by gel electrophoresis nor that by steady‐state electrofocusing seems sufficiently convenient. By contrast, transient state electrofocusing provides a ready means for their separation.
A measuring grid for the analysis of isoelectrically focused proteins in polyacrylamide gel slabs
Authors: Gassner George , Sears Sheila , Graham Charles , Janardan Konanur G.
Published: May 2005

An overlay grid was constructed to facilitate the measurement of densiometric scans of proteins in pH gradients formed by electrofocusing. The overlay procedure provides a rapid way for data collection. The relationship between pH and gel distance closely fit a straight line, (R>0.99). The reliability of the system was shown by the close agreement of the linear regressions from six different sets of data. Also, the feasibility of using a similar grid for a nonlinear system was established.
Two‐Dimensional gel electrophoresis of dexamethasoneinduced organ‐specific developmental proteins (fsa,fsb) in rat lung fibroblasts
Authors: Floros Joanna , Phelps David S. , Smith Barry T.
Published: May 2005

Glucocorticoids are known to enhance fetal lung maturation. Previous studies with whole lung have shown that specific proteins are enhanced by these hormones. At least two effects of glucocorticoids on fetal lung epithelia require the presence of mesenchymal elements. Since the lung consists of several different cell types, further understanding of glucocorticoid effects on fetal lung protein synthesis requires that such studies be carried out with specific cell types. Using two‐dimensional gel electrophoresis, we have examined the synthesis and secretion of proteins by fetal rat lung fibroblasts in the presence and absence of glucocorticoid. Two proteins, fsa and fsb, are enhanced by dexamethasone, appear to be organ specific, and are enhanced only during prenatal and early postnatal life. They are not enhanced by a variety of other hormones.
Mathematical simulation of steady state isoelectric focusing of proteins using carrier ampholytes
Authors: Shimao Kazuo
Published: May 2005

Steady state isoelectric focusing was treated as zero velocity isotachophoresis. From this standpoint, the author's theory of isotachophoresis was modified to deal with isoelectric focusing of protein molecules using carrier ampholytes, and mathematic simulations according to the modified theory were performed to calculate pH, voltag and concentration profiles in steady state isoelectric focusing. The results were in agreement with experiments with regard to resolving power for protein separation.
A new method for sex determination of the donor of forensic samples using a recombinant DNA probe
Authors: Gill Peter
Published: May 2005

Microscopical methods for sex determination of denatured samples in forensic laboratories are frequently unreliable. This paper describes the use of a recombinant DNA probe which hybridises specifically to a 2.47 kb repeat sequence of which 2000 copies are present on the Y chromosome. The technique enables rapid and reliable sex determinations of degraded DNA samples and produces results from blood stains greater than 4 years old in which only low molecular weight (< 10 kb) DNA is present. Stains from less than 1 μL of blood or a single hair root is required for the test using dot blot hybridisations. Electrophoretic analyses revealed that Hae III restriction of DNA samples produced somatically stable patterns.
Horizontal two‐dimensional electrophoresis with immobilized pH gradients in the first dimension in the presence of nonionic detergent
Authors: Görg Angelika , Postel Wilhelm , Weser Johann , Günther Siegfried , Strahler John R. , Hanash Samir M. , Somerlot Luke
Published: May 2005

Horizontal two‐dimensional (2‐D) electrophoresis with immobilized pH gradients (IPG) in the first dimension, described for soluble proteins in Electrophoresis 1985, 6, 599–604, has been extended to an analysis of complex protein mixtures, such as leukemia cell proteins or bean proteins, in the presence of nonionic detergents. By optimizing the ratio of gel volumes for the first and second dimensional separation, and by decreasing the concentration of Nonidet P‐40 in the IPG gel, undistorted protein patterns in the 2‐D gel are obtained. Horizontal and vertical streaking are considerably diminished by optimized reswelling conditions of the dry IPG strips in presence of both urea and detergent. Resolution is improved and a reduced background is obtained when overcrowded gel areas are spread by flattening the pH gradient (in the first dimension) and the polyacrylamide gradient (in the second dimension).
Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes
Authors: Altland Klaus , von Eckardstein Arnold , Banzhoff Angelika , Wagner Michael , Rossmann Ute , Hackler Rolf , Becher Pia
Published: May 2005

Comparing the separation patterns achieved by hybrid isoelectric focusing in immobilized pH gradients of 1 and 3 meqv/pH/L buffering power, it is demonstrated that a significant proportion of sample protein is lost in gels with the higher buffering power. A buffering power of 1 meqv/pH/L was found to be the lowest acceptable limit for flat bed gel hybrid isoelectric focusing still ensuring adequate control of the immobilized pH gradient. Protein losses were manifested as diminished overall staining intensity, disappearance of individual zones in dilution series, tailing effects and residual material at the application site, all of the above effects being more pronounced for larger than smaller proteins. Adsorption of proteins to charged binding sites of the immobilized pH gradient offers a plausible explanation for the observed phenomena. The visible effects of adsorption could be reduced by the addition of carrier ampholytes, but not by urea or detergents like Triton X‐100, 3‐(3‐chol‐amidopropyl)‐dimethylammonio‐l‐propanesulfonate (CHAPS) or octyl β‐thio‐glucoside, indicating ionic interactions between the immobilized charged groups, carrier ampholytes and proteins. The adsorption phenomena may strongly affect the quantification of proteins in immobilized pH gradients.
Two‐dimensional lectin affinity electrophoresis of α‐fetoprotein: Characterization of erythroagglutinating phytohemagglutinin‐dependent microheterogeneity forms
Authors: Taketa Kazuhisa , Ichikawa Eriko , Sato Jiro , Taga Hiroko , Hirai Hidematsu
Published: May 2005

By means of two‐dimensional lectin affinity electrophoresis of human α‐fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E‐PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP‐P2 (E‐PHA‐nonreactive) but also AFP‐P4 and P5 (E‐PHA‐reactive) had affinities for Concanavalin A (Con A) and Allomyrina dichotoma lectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex‐type oligosaccharides with the terminal galactose on Man α1 → 6 residue sialylated at the C‐6, but not C‐3, position. On the other hand, the results with a hepatoblastoma (HUH‐6 C1–5 cell line) AFP showed that not only AFP‐P5 but also AFP‐P1 (E‐PHA‐nonreactive) and P3 (E‐PHA‐less reactive) had Con A‐nonreactive AFP and that AFP‐P1 had AFP‐A1 (allo A‐nonreactive) and AFP‐A2 (allo A‐less reactive), and AFP‐P3 and P4 had AFP‐A1s (allo A‐nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E‐PHA‐dependent bands of AFP were further subdivided with Lens culinaris agglutinin (LCA‐A) into LCA‐A‐reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocullular carcinomas and other malignancies, indicating that the bisected bi‐(or tri‐and tetra‐) antennary sugar chains with the exposed terminal galactose of the Man α 1 → 6 arm as well as those with the C‐3 sialylated galactose residues could be expressed in AFP upon malignant transformation. The two‐dimensional lectin affinity electrophoresis thus proves useful for further characterization of lectin‐dependent AFP bands.
Crossed affino‐immunoelectrophoresis or affino‐blotting with lectins: Advantages and limitations for glycoprotein studies
Authors: Faye Loïc , Salier Jean‐Philippe
Published: May 2005

In contrast to the conventional combination of physical, chemical and enzymatic methods used for a structural analysis of glycans in glycoproteins, alternative methods involve affinity electrophoresis as a tool for the detection, characterization, and quantitation of glycoproteins and their carbohydrate moiety, owing to interactions with lectins. Two major approaches involve (i) crossed affino‐immunoelectrophoresis and variations thereof, whereby lectin/glycoprotein interactions occur during the electrophoretic runs, or (ii) affino‐blotting, where the glycoproteins are electrophoretically separated and then immobilized onto a solid support prior to their interaction with lectins. A critical comparison of these two series of techniques is the scope of the present paper. These techniques are of high interest by virtue of their ability at differentiating a classical glycan structure from unusual oligosaccharide side chains. The former structures will usually be qualitatively and quantitatively described with the easy and fast procedures as well as the simple equipment required for crossed affino‐immunoelectrophoresis or affino‐blotting, whereas the latter will be good candidates for further structural analyses.
Enzyme activity electrophoresis: Development and applications
Authors: Poulsen Otto M. , Jacobsen Tomas , Hau Jann , Jensen Bo , Vodder Lise , Andersen Frantz
Published: May 2005

The development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme‐substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studies.
Free‐flow electrophoresis under microgravity: Evidence for enhanced resolution of cell separation
Authors: Hannig Kurt , Kowalski Matthias , Klöck Gerd , Zimmermann Ulrich , Mang Volker
Published: May 2005

A mixture of fixed rabbit, guinea pig and rat erythocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g ‐ and μg ‐ conditions using a specially designed electrophoretic module. Short duration μg conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g‐conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g‐conditions, clear separation of the cell mixture could be recorded after lift‐off of the rocket under μg‐conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire μg‐phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under μg‐conditions in media of high ionic strength.
Electrophoretic transport of solutes in aqueous two‐phase systems
Authors: Levine Mark L. , Bier Milan
Published: May 2005

Electrophoretic transport of proteins across the interface between the phases of an aqueous polymer two‐phase system can be greatly impeded in comparison with transport within the individual phase. This effect can be controlled by modifying the affinity of the protein for a phase by suitable manipulations of such variables as pH. The effect is not caused by differences in the electrophoretic velocity between the two phases, nor by large changes in pH at the interface. An analogy exists between this phenomenon and the related subject of diffusion of electrolytes across the phase interface.
Enzyme visualization with partially dehydrated agarose substrate layers: Detection of paraoxonase after thin‐layer isoelectric focusing in agarose
Authors: Haas Arved , Mallinckrodt Marika Geldmacher‐Von
Published: May 2005

A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3‐naphtyl acetate, paraoxon, 5‐bromo‐4‐chloro‐3‐indolyl acetate and 5‐bromo‐4‐chloro‐3‐indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA‐stable. Immunofication with anti‐human serum albumin‐antibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and /or cleavage products is the major problem.
Resolution of a paradox in the electrophoresis of DNA in agarose gels
Authors: Stellwagen Nancy C. , Holmes Diana L.
Published: May 2005

A paradox was observed in a previous study of the electrophoresis of linear DNA fragments in agarose gels (D. L. Holmes and N. C. Stellwagen, Electrophoresis 1990, 11, 5–15). The pore size of the agarose matrix was more accurately determined if the root‐mean‐square radius of gyration was used to measure DNA macromolecular size. However, the Ogston equations were obeyed and other gel parameters such as the apparent fiber radius and fiber volume appeared to be better described if the geometric mean radius was used to measure DNA size. This paradox can be resolved if relative mobilities (with respect to the smallest DNA molecule in the data set) are used to construct the Ferguson piots, instead of absolute mobilities. Using relative mobilities and the root‐mean‐square radius of gyration, the Ogston equations are obeyed and the pore size of the matrix is consistent with values determined by other methods.
Two‐dimensional gel protein database of Saccharomyces cerevisiae
Authors: Boucherie Hélian , Sagliocco Francis , Joubert Richard , Maillet Isabelle , Labarre Jean , Perrot Michel
Published: May 2005

With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two‐dimensional (2‐D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2‐D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N‐terminal acetylated proteins, mitochondrial proteins, glucose‐repressed proteins, heat shock induced proteins and proteins encoded by intron‐containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.
cDNA expression and human two‐dimensional gel protein databases: Towards integrating DNA and protein information
Authors: Leffers Henrik , Dejgaard Kurt , Honoré Bent , Madsen Peder , Nielsen Morten S. , Celis Julio E.
Published: May 2005

The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two‐dimensional (2‐D) gel electrophoresis in combination with computer‐aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To data, comprehensive 2‐D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http://biobase.dk/cgi‐bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2‐D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post‐translational trimmings and modifications of the proteins. The method, in combination with 2‐D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.
Analysis of chaperonin‐containing TCP‐1 subunits in the human keratinocyte two‐dimensional protein database: Further characterisation of antibodies to individual subunits
Authors: Hynes Gillian , Celis Julio E. , Lewis Victoria A. , Carne Alexander , U Sally , Lauridsen Jette B. , Willison Keith R.
Published: May 2005

The chaperonin‐containing TCP‐1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two‐dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP‐1, also known as CCTα, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP‐1, and a number of epitope‐related proteins were identified.
The use of two‐dimensional gel electrophoresis in studies on the role of cytoskeletal plaque proteins as tumor suppressors
Authors: Ben‐Ze'ev Avri
Published: May 2005

The morphology and functions of cell sand tissues are determined, in a large part, by mechanical forces generated at cell‐cell and cell‐extracellular matrix (ECM) contacts. At these sites, transmembrane adhesion receptors of the integrin and cadherin families are linked, via their cytoplasmic domain, to the cytoskeleton by submembranal plaque proteins such as vinculin, α‐actinin and the cell‐cell junctional plaque proteins α‐ and β‐catenin and plakoglobin (or γ‐catenin). Recent studies have implicated this link of structural molecules between the outside and inside of the cell in signal transduction. We have shown that the expression of junctional plaque proteins is modulated during growth stimulation and differentiation, and is dramatically reduced in certain tumor cells. To study the functional significance of these changes in expression, we have used recombinant DNA technologies to overexpress or suppress the levels of junctional plaque proteins. In addition, we eliminated the expression of vinculin in embryonal stem (ES) cells and in the embryonal carcinoma F9 line by gene disruption employing homologous recombination. The results have indicated that moderate overexpression of cell‐ECM plaque proteins results in reduced cell motility. In contrast, suppression of their expression, by antisense transfection, led to enhanced motility and conferred anchorage independent growth and tumorigenicity, upon injection into nude mice. These findings suggest that submembranal plaque proteins can act as effective tumor suppressors. In agreement with this notion, we found in several tumor cell lines diminished levels of junctional plaque proteins. Restoration of their level to that found in normal cells resulted in tumor suppression after their injection into experimental animals. Here we demonstrate the usefulness of the application of two dimensional (2‐D) gel electrophoresis in these studies.
Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications
Authors: Wätzig Hermann , Degenhardt Matthias , Kunkel Annette
Published: May 2005

This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electrosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.