Gel electrophoresis as a nanoseparation tool serving DNA nanotechnology

The past years have witnessed a rapid development of DNA nanotechnology in nanomaterials science with a central focus on programmable material construction on the nanoscale. An efficient method is therefore highly desirable (but challenging) for analytical/preparative purification of DNA-conjugated nano-objects and their DNA-assemblies. In this regard, agarose gel electrophoresis, a traditional technique that has been invented for biomacromolecule separation, has found many innovative uses. This includes shape, size, charge, and ligand-valence separations of nanoparticle building blocks as well as monitoring a self-assembly process towards product identification and purification.     

Chiral separation based on ligand-exchange capillary electrophoresis using a copper(II)-L-ornithine ternary complex as selector

A ligand-exchange capillary electrophoresis was explored, with L-ornithine as the ligand and copper(II) as the central ion. Its applicability was demonstrated with underivatized and dansyl amino acids, a dipeptide, and drugs with amino alcohol structure. The enantioselectivity was found to be strongly dependent on pH and copper(II)-L-Orn complex concentration. Due to the adsorption of the positively charged species onto the capillary inner walls, the chiral separation selectivity is very high while the efficiency is relatively low. Permanent 1,3-propanediamine-coated capillaries show an improved separation efficiency and theoretical plate numbers increasing from 10(4) to 10(5). Similar phenomena were observed when sodium dodecyl sulfate (SDS) micelles were added to the copper(II) complex solution. The poor separation efficiency of chiral compounds in uncoated capillaries may result from the low rate of the ligand-exchange reactions, and the high enantioselectivity may derive from the complexing process in the adsorbed phase.     

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.     

Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.     

Complexometric titrations by means of stepwise complex formation

Summary A review is given of the possibility to use stepwise complex formation for complexometric titrations. The selectivity of the determinations of metal ions, the limits of determination as well as the required conditions for the stability constants are discussed.

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Komplexometrische Titrationen mit stufenweiser Komplexbildung

Zusammenfassung Eine Übersicht wird gegeben über die Möglichkeit, stufenweise Komplexbildung für komplexometrische Titrationen auszunützen. Die Selektivität der Bestimmungen von Metallionen, die Bestimmungsgrenze sowie die Bedingungen für die Werte der Stabilitätskonstanten werden diskutiert.

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Dedicated to Prof. Dr. H. Weisz on occasion of his 60th birthday     

Thermodynamics of complex formation in ternary liquid mixtures containing acetonitrile

Vapor—liquid and liquid—liquid equilibria and excess enthalpies for ternary mixtures formed from acetonitrile, benzene, n-heptane, toluene, and carbon tetrachloride are successfully correlated with a modified version of the associated solution theory proposed by Lorimer and Jones in 1977, which assumes two types of self-association for acetonitrile and binary complexes between acetonitrile and unsaturated hydrocarbons and does not include any ternary parameters.     

Enhancement of chiral recognition by formation of a sandwiched complex in capillary electrophoresis

For chiral primary amino compounds not separable by cyclodextrins alone, chiral recognition was successfully achieved by the formation of a sandwiched complex of the non-chiral 18-crown-6, the chiral amine and cyclodextrin (CD) [18-crown-6+amino compound+CD]. The separation of 1-methyl-3-phenylpropylamine and 1,2,3,4-tetrahydro-1-naphthylamine racemates showed the special function of the non-chiral 18-crown-6 on chiral recognition. By formation of the sandwiched complex, the chiral center of 1-methyl-3-phenylpropylamine was successfully recognized, and resolution of 1,2,3,4-tetrahydro-1-naphthylamine dramatically increased. In these studies, the mobility differences of the enantiomers were evaluated as a function of the concentration of cyclodextrins with and without the 18-crown-6, and as a function of the concentration of the 18-crown-6. In addition, the separations by this method were compared to those by the chiral 18-crown-6 reagent.     

The cyclodextrin-nicotinamide compound as a dehydrogenase model simulating apoenzyme-coenzyme-substrate ternary complex system

The cyclodextrin-dihydronicotinamde had a dihydronicotinamde group at the open side of cyclodextrin cavity, and showed a large rate enhancement in the reduction of substrate upon complexation comparing with NADH.     

Spectrophotometric determination of maneb by ternary complex formation with PAR and CTAB

A rapid, simple, direct, and sensitive method has been developed for the determination of maneb (manganese ethylenebisdithiocarbamate) based on the formation of manganese-4-(2′-pyridylazo) resorcinol complex by a ligand displacement reaction, which is rendered water soluble by a cationic surfactant cetyltrimethylammonium bromide (CTAB) by the formation of an ion association complex. Beer's law is obeyed over the concentration range 0.08–2.4 μg/ml of the final solution at 500 nm in pH range 8–12. The molar absorptivity and Sandell's sensitivity are calculated to be 8.84 × 10⁴ l.mol⁻¹.cm⁻¹ and 0.003 μg/cm², respectively. The developed method has been applied to the determination of maneb in commercial formulations, synthetic mixture, grain samples and vegetables.     

The spectrophotometric determination of thallium(iii)by ternary complex formation

If thallium(III) is added to an aqueous solution of potassium thiocyanate containing a large amount of pyridine in the pH range 5.2–5.5, a yellow solution which is stable in diffuse light is obtained. The yellow colour can be measured at 405 nm for the colorimetric determination of thallium(III) in the range 20–300 μg Tl ml^-1. The complex is a mixed ligand complex with a metal-ligand ratio of 1:2:2. Thallium(I) does not interfere. The interference of various other metal ions and anions is discussed.     

Syntheses of some analogs of a possible intermediate formed in the thymidylate synthetase reaction

The syntheses of a number of analogs of a possible intermediate formed in the thymidylate synthetase reaction are described. These consist of a number of 5-arylaminomethyluracils (X) and N-methyl-N-[1,-(2′-deoxy-β-D-ribofuranosyl)thyminyl]-p-aminobenzoyl glutamic acid (XXb).     

Activation of chlorobenzene as phenylating agent by means of a dinitrogen-molybdenum complex

Bis(dinitrogen)bis[1,2-bis(diphenylphosphino)ethane]molybdenum reacts with chlorobenzene to form molybdenum chloro complexes and phenylated organic products. Benzene, biphenyl, o-, m-, p-chlorobiphenyl, a dihydro derivative of o-chlorobiphenyl, triphenylphosphine and diphenylvinylphosphine were formed. The isomer distribution of the chlorobiphenyls is close to that obtained by decomposition of 0.02 M benzoyl peroxide in chlorobenzene under dinitrogen. The cleavage of the PhCl bond induced by the molybdenum-dinitrogen complex appears to give rise to the same homolytic reaction pattern. Phenylation of anisole and of triethyl phosphite has also been achieved with the same reagent.     

Chelate complex formation as a basis for titration processes

Metal cations can be looked upon as LEWIS acids and their reaction with ligands during complex formation can be compared to the addition of the proton to a base. The complex formation reactions, however, lack sharp and definite endpoints and metal cations therefore cannot be titrated with ammonia, cyanide and other ligands in the same way as the hydrogen ion can be titrated alkalimetrically. The theoretical reason for this fact is discussed and it is shown that complex formations gain the characteristic properties of neutralisation reactions if the simple ligands are replaced by a polydentate group which is able to satisfy not only one, but several of the coordination points of the metal cation. A great number of titration processes can be based on such reactions.     

Conductometric titration of nitrate by formation of the silver-1,10-phenanthroline-nitrate ternary complex

Nitrate forms an insoluble ternary complex, Ag(phen)(2)NO(3), with silver in the presence of 1,10-phenanthroline (phen). A simple and direct conductometric titration of nitrate, based on the formation of this ternary complex, has been investigated. The sample solution is adjusted to pH 4.3 with acetate buffer and titrated with 0.1M Ag(phen)(+)(2) complex solution in 40% ethanol-water mixture. The relative error is less than 0.5% and relative standard deviation less than 0.4%. The effect of ammonium ion and urea has also been studied. The method has been successfully applied to determination of nitrate-nitrogen in fertilizer.     

Determination of citric acid by means of competitive complex formation in a flow injection system

A photometric method for the determination of citrate and other organic acids based on their ability to complex Fe³⁺-ions is presented. The red colored complex of [Fe(SCN)₂]⁺, used as reagent, is destroyed upon contact with the sample because the organic acid complexes the Fe³⁺-ion. The decrease in absorption is monitored at 460 nm. The reaction is carried out in a simple flow injection system either in single or preferably double channel configuration.The influence of pH was investigated. Best results were obtained by adjusting the carrier stream to pH 2.0–2.5 with a KCl/HCl-buffer. With an increasing concentration of reagent the linear range is shifted to higher citrate concentrations. The slope of the calibration graph and the linear range are influenced by the sample volume. Other variations of parameters include flow rate, reactor volume and diameter of tubing. Generally speaking, optimum conditions for the flow system are not specified because they vary with the application.The typical conditions for a calibration graph from 1 to 8 mmol/l citrate were a reagent concentration of 2.6 mmol/l [Fe(SCN)₂]⁺, a flow rate of 2.4 ml/ min, a reactor length of 50 cm with tubing of 0.97 mm inner diameter and a sample volume of 100 l. At these system settings the coefficients of variation were 2.5% and 1.6% for eight replicate measurements of samples containing 4 mmol/l and 8 mmol/l citrate, respectively. Up to 180 samples can be analyzed per hour.Naturally the method is disturbed by all other ions that form complexes or precipitates with Fe³⁺-ions. Therefore its application is limited to samples with a known matrix, which was given in the analysis of citrate in lemon flavored soft drinks, where the citric acid usually accounts for 95 to 99% of the total acidity and other interfering ions are absent.     

Fluorimetric determination of magnesium by ternary complex formation with pyridoxal nicotinylhydrazone and amines

The synthesis, characteristics and analytical applications of pyridoxal nicotinylhydrazone are described. This compound reacts with magnesium(II) in the presence of ammonia, ethylenediamine or pyridine, to produce a 1:1:1 Mg(II)—pyridoxal nicotinylhydrazone—amine fluorescent complex (λ_ex 395 nm, λ_em 480 nm). A fluorimetric method is proposed for the determination of magnesium(II) (20–100 ng ml^-1 in the solution measured); isobutanol is used to extract the complex, reducing the number of interferences.     

Fluorimetric determination of sulphate by ternary complex formation with zirconium and biacetylmonoxime nicotinylhydrazone

A fluorimetric procedure for the determination of sulphate (0.5-6 mug ml ) based on the formation of a ternary complex with biacetylmonoxime nicotinylhydrazone and zirconium(IV) is described. The excitation and fluorescence maxima are at 415 and 505 nm, respectively. The method is rapid and the procedure reproducible. The precision is +/- 2% near to the middle range of the calibration curves. There is no interference from most common ions. Phosphate and fluoride are tolerated at 100-fold molar ratio to sulphate. The method has been applied to the determination of sulphate in a variety of water samples.     

Combinatorial dispensing as a fast and efficient means to create complex screens

Liquid handling robots carry out tasks from simple plate filling to complex operations such as creating reagent cocktails from multiple stock solutions. The latter task is conceptually a combinatorial process where each cocktail is created by combining a subset of stock solutions in user-defined volumes. General-purpose liquid handlers can perform this task, but their hardware lacks the inherent properties needed to exploit the combinatorial nature of the problem at hand. Here we present the use of non-contact dispensing technologies to create complex screens at low volume and high density. Our approach is based on the "inkjet printer principle" where a block of dispensers (print head) travels over a multi-well plate (paper) to deliver the reagents (inks) in a user-defined pattern. Impact-induced mixing and the lack of tip contamination remove the need for extensive tip washing or the use of large numbers of disposable tips. As an example, protein crystallization screening is used to demonstrate the technology. This application requires the creation of complex mixtures from many stock solutions with a great diversity of viscosities and surface tensions. In addition, dispense volumes cover a range from 50 nL to 50 microL, illustrating its utility in low-volume high-density screening.