High-Voltage Paper Electrophoresis (HVPE).

HVPE is an excellent and often overlooked method for obtaining objective and meaningful information about cell-wall "building blocks" and their metabolic precursors. It provides not only a means of analysis of known compounds but also an insight into the charge and/or mass of any unfamiliar compounds that may be encountered. It can be used preparatively or analytically. It can achieve either "class separations" (e.g., delivering all hexose monophosphates into a single pool) or the resolution of different compounds within a given class (e.g., ADP-Glc from UDP-Glc; or GlcA from GalA).All information from HVPE about charge and mass can be obtained on minute traces of analytes, especially those that have been radiolabeled, for example by in-vivo feeding of a 3H- or 14C-labeled precursor. HVPE does not usually damage the substance under investigation (unless staining is used), so samples of interest can be eluted intact from the paper ready for further analysis. Although HVPE is a technique that has been available for several decades, recently it has tended to be sidelined, possible because the apparatus is not widely available. Interested scientists are invited to contact the author about the possibility of accessing the Edinburgh apparatus.

Low-voltage origami-paper-based electrophoretic device for rapid protein separation.

We present an origami paper-based electrophoretic device (oPAD-Ep) that achieves rapid (∼5 min) separation of fluorescent molecules and proteins. Due to the innovative design, the required driving voltage is just ∼10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep uses multiple, thin (180 µm/layer) folded paper layers as the supporting medium for electrophoresis. This approach significantly shortens the distance between the anode and cathode, and this, in turn, accounts for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep enables convenient sample introduction by use of a slip layer as well as easy product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. We demonstrate the use of oPAD-Ep for simple separation of proteins in bovine serum, which illustrates its potential applications for point-of-care diagnostic testing.

Preparation and properties of electrophoretic microcapsules for electronic paper.

This paper shows two types of microcapsules used for electrophoretic display. One is prepared by in-situ polymerization which is based on urea, melamine and formaldehyde and another by complex coacervation, which is composed of gelatin and gum Arabic. Microcapsules attract interests of many research groups for longer lifetime of electrophoretic display by reducing agglomerization or lateral movements of nanoparticles. The gelatin microcapsules were more attractive in providing more uniform microcapsule coverage on electrodes due to their flexibility as compared to the melamine-urea microcapsules. The properties of microcapsules were characterized by FTIR, OM, SEM and TGA. Migration of nanoparticles in the two types of microcapsules was also observed when an electric field was applied.

Paper electrophoretic determination of the stability constants of binary and ternary complexes of copper(II) and cobalt(II) with nitrilotriacetate and cysteine.

A paper ionophoretic method is described for the study of equilibria in mixed ligand (nitrilotriacetate-cysteine) complex system in solution. The proportion of ionic species of nitrilotriacetate (NTA) and cysteine were varied by changing the pH of background electrolyte. The stability constants of Cu(II)-NTA-cysteine and Co(II)-NTA-cysteine complexes were found to be 6.35+/-0.05 and 5.45+/-0.02 (logarithm stability constant values), respectively, at ionic strength 0.1 M and a temperature of 35 degrees C.

A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase.

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver.

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.

Purification of anthocyanins from species of Banksia and Acacia using high-voltage paper electrophoresis.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography-electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives.

Electrophoretic studies on the chelating tendency of bioactive sulphur-containing amino acids. The metal-methylcysteine-cysteine system.

Stability constants of binary Fe(III)-methylcysteine, Cr(III)-methylcysteine and mixed Fe(III)-methylcysteine-cysteine, Cr(III)-methylcysteine-cysteine complexes have been determined by paper electrophoresis at 0.1 M ionic strength and a temperature of 35 degrees C. The stability constants of Fe(III)-methylcysteine-cysteine and Cr(III)-methylcysteine-cysteine mixed complexes were found to be 6.00 +/- 0.07 and 5.05 +/- 0.15 (logarithm of stability constant values), respectively.

From paper electrophoresis to computer-supported interpretation of capillary electrophoresis--clinical plasma protein analysis in Malmö, Sweden.

Protein analyses have been used in Malmö as a routine clinical diagnostic tool since 1953. Most serum samples are submitted for "protein profiles" including capillary zone electrophoresis and rate immune nephelometric quantification of nine proteins (five in urines), although analysis of single proteins may be requested. Standardization between laboratories in our region has been greatly improved by automation, CRM 470 calibration and external quality assurance. We are further extending standardization by developing computer supported interpretations using a program with improved user interface and graphical representation of electrophoretic curves superimposed upon a shaded reference interval. Programming is underway to provide complete automatic interpretation of these curves. Together, capillary electrophoresis (with access to mathematical analysis) and immunochemical quantifications allow a highly automated process accessible to further digital analysis and automated interpretation. Rapid, cost-effective and standardized analysis of serum protein profiles should improve the diagnostic evaluation of many categories of patients.

[The use of electrophoretic methods for determining modified proteins in diabetic patients]. / Primenenie élektroforeticheskikh metodov dlia opredeleniia modifitsirovannykh belkov u bol'nykh sakharnym diabetom.

Modified proteins were determined by isoelectric focusing in borate-polyol system with subsequent colorimetry (micromethod) and electrophoresis of blood serum on paper with subsequent TCA-ethanol treatment. Increased levels of glycated hemoglobin and modified albumin and changed light absorbance of glycated albumin were detected. The levels of glycated hemoglobin assessed by the micromethod and colorimetry without calibration did not correlate.

New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs.

In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.

Paper electrophoresis in the study of mixed ligand complexes in solution. The system Fe(III)- and Cr(III)-methyl cysteine-penicillamine.

A method involving use of paper electrophoresis is described for the study of equilibria in mixed ligand [methyl cysteine-penicillamine] complex systems in solution. The concentration of the primary ligand methyl cysteine was kept constant, while that of secondary ligand penicillamine was varied. The plots of -log [penicillamine] against mobility were used to obtain information on the formation of mixed complex and to calculate its stability constants. The binary equilibria M(III)-methyl cysteine have also been studied since this is a pre-requisite for the investigation of mixed complexes. The stability constants of [Fe(III)- methyl cysteine-penicillamine] and [Cr(III)-methyl cysteine-penicillamine] complexes were found to be 7.40 +/- 0.13 and 4.80 +/- 0.07 (log K values) for Fe(III) and Cr(III) complexes, respectively, at mu = 0.1 mol/L and at a temperature of 35 degrees C.

Attempts to identify the active factor of reticulin-M by high voltage electrophoresis and by acetone precipitation.

The reticulin-M forte (R forte), an antianaphylactic peptide, extracted from organs rich in RES, previously stimulated with India ink, was analysed after acetone precipitation by paper high voltage electrophoresis. Finally the biological activity remains concentrated in the second, arbitrary group of basic peptides, fractions 1 and 3.

Asparagine-linked sugar chains of plasma membrane glycoproteins from healthy and common variable immunodeficiency B lymphoblastoid cell lines.

Asparagine-linked sugar chains of plasma membrane glycoproteins, which are formed by glycosylation during B cell maturation, were examined with B lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus derived from healthy controls and patients with common variable immunodeficiency (CVI). Both two patients with CVI showed hypogammaglobulinemia and impaired B cell functions. LCLs from healthy controls and the patients showed CD19+ and HLA/DR+ in the cell surface and secreted IgM. In both healthy controls and the patients, the main oligosaccharide in asparagine-linked sugar chains of the membrane glycoproteins of LCLs was biantennary sugar chain with bisected GlcNAc (Gal2-GlcNAc2-Man3-GlcNAc-GlcNAc-Fuc-GlcNAcOT). Biantennary sugar chain with an alpha-fucosyl residue linked at the proximal GlcNAc was seen but biantennary sugar chain without an alpha-fucosyl residue at the proximal GlcNAc was little detected in each LCL. There was no difference in quality and quantity of asparagine-linked sugar chains between healthy controls and the patients. These results suggest that glycosylation during B cell maturation may not be impaired in patients with CVI.

A competitive radioimmunoassay for peptidoglycan monomer.

The preparation and characterization of the essential components to be used in the radioimmunoassay of peptidoglycan monomer (PGM) is described. In order to raise the anti-peptidoglycan monomer antibodies 14C-labelled peptidoglycan monomer-bovine serum albumin conjugate was prepared by the coupling of 14C-peptidoglycan monomer to bovine serum albumin in the presence of glutaraldehyde in 0.1 M NaHCO3 at pH 8.3. The prepared conjugate elicited anti-PGM response in rabbits. A synthetic analog of peptidoglycan monomer, Boc-L-tyrosyl-peptidoglycan monomer was prepared by condensation of unprotected peptidoglycan monomer and N-hydroxysuccinimidester of Boc-L-tyrosine in the presence of triethylamine and the obtained disaccharide-hexapeptide was labelled with Na125I. This compound exhibited the ability of binding to anti-peptidoglycan monomer antibodies. The prepared compounds, namely anti-PGM antibodies and 125I-labelled Boc-L-tyrosyl-peptidoglycan monomer, were used as essential components in competitive radioimmunoassay for peptidoglycan monomer determination in mammalian and human sera and plasma, respectively.

Aminoacidos urinarios: electroforesis por alto voltaje y acetato de celulosa gelatinizado / Urinary amino acids: electrophoresis for high voltage and in gelatinized cellulose acetate

El estudio de las alteraciones del metabolismo de los aminoacidos urinarios ha motivado el desarrollo de dos tecnicas simples, rapidas y economicas, para el uso en Quimica clinica. Se describe la celda electroforetica disenada, asi como todas las operaciones requeridas para lograr dicho objetivo (tratamiento de los soportes usados y de las muestras, buffers, condiciones de corrida, distintos metodos y reactivos de revelado, desintometria). Se comparan los resultados obtenidos para los soportes empleados, papel y acetato de celulosa gelatinizado, usando para ambos igual sistema de refrigeracion y los resultados obtenidos en comparacion con otra forma de enfriamiento del sistema, utilizada previamente. Tambien se discute la aplicacion de mayores voltajes y la importancia de la estandarizacion en la coloracion. Trabajando con acetato de celulosa gelatinizado se pueden valorar por densitometria las corridas, lo cual brinda una util herramienta a los laboratorios clinicos

Identification of covalently bound fatty acids on acylated proteins immobilized on nitrocellulose paper.

A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.