Use of anion-exchange high-performance liquid chromatography for the study of smooth muscle myosin light-chain kinase and its catalytic domain

Avian myosin light-chain kinase from smooth muscle of the gizzard and its catalytic domain, derived from the intact enzyme by trypsin digestion, was purified within 30-40 min by both analytical and preparative anion-exchange high-performance liquid chromatography. The proteins obtained were more than 95% pure and retained their biological activity. The high-performance anion-exchange chromatography protocols represent a significant decrease in purification time when compared with conventional ion-exchange chromatography.     

Complete systems, elementary submodels and the tightness of upper hyperspaces

We introduce and study some completeness properties for systems of open coverings of a given topological space. A Hausdorff space admitting a system of cardinality κ satisfying one of these properties is of type Gκ. Hence, we define several new variants of the ?ech number and use elementary submodels to determine further results. We introduce M-hulls and M-networks, for M elementary submodel. As an application, we give estimates for both the tightness and the Lindelöf number of a generic upper hyperspace. Two recent results of Costantini, Holá and Vitolo on the tightness of co-compact hyperspaces follow from ours as corollaries.     

Weighted approximation of functions on the real line by Bernstein polynomials

The authors give error estimates, a Voronovskaya-type relation, strong converse results and saturation for the weighted approximation of functions on the real line with Freud weights by Bernstein-type operators.     

In situ preparation of protein-"smart" polymer conjugates with retention of bioactivity

Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by muLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by (1)H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed.     

Anodic stripping voltammetry of sulphide at a nickel film: towards the development of a reagentless sensor

The determination of sulphide at an electrochemically generated nickel oxide layer at glassy carbon and screen-printed electrodes in acidic media has been examined and appraised. The NiO layer was found to produce a stripping-like signal to sulphide and gave a linear peak current response from 20 to 90 μM. The response was further enhanced by repetitive cycling allowing accumulation of nickel sulphide at the electrode surface such that lower micromolar levels of sulphide (i.e. 5 μM) can be determined. The response at the NiO layer to sulphide is shown to be reproducible over a period of 24 h, thereby offering the development of a disposable amperometric sensor for sulphide.     

Determination of ammonia based on the electro-oxidation of hydroquinone in dimethylformamide or in the room temperature ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

The results detail a novel methodology for the electrochemical determination of ammonia based on its interaction with hydroquinone in DMF. It has been shown that ammonia reversibly removes protons from the hydroquinone molecules, thus facilitating the oxidative process with the emergence of a new wave at less positive potentials. The analytical utility of the proposed methodology has been examined with a linear range from 10 to 95 ppm and corresponding limit-of-detection of 4.2 ppm achievable. Finally, the response of hydroquinone in the presence of ammonia has been examined in the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluormethylsulfonyl)imide, [EMIM][N(Tf)₂]. Analogous voltammetric waveshapes to that observed in DMF were obtained, thereby confirming the viability of the method in either DMF or [EMIM][N(Tf)₂] as solvent.     

Verminderung der Einwaage bei der Schwefelmikrobestimmung nach Schöniger

Zusammenfassung Es wurde die Möglichkeit untersucht, die Probemenge bei der Schwefelmikrobestimmung nachSchöniger, ohne das gewöhnliche Verfahren wesentlich zu modifizieren, durch Anwendung eines geänderten Kolbens zu vermindern.Innerhalb der üblichen Toleranzgrenzen liegende Ergebnisse (Standardabweichung ± 0,13%) wurden mit Einwaagen von 1,5 bis 2 mg erhalten.

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Summary The possibility was investigated of reducing the weight of sample in the micro determination of sulfur by theSchöniger procedure, but without essentially modifying the usual method, namely by using a modified flask. Samples weighing 1.5 to 2 mg gave results within the usual tolerance limits (standard deviation ± 0.13%).

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Résumé On a étudié la possibilité de diminuer la prise d'essai dans le mierodosage du soufre suivantSchöniger, sans altérer d'une manière importante le procédé habituel, en utilisant un ballon modifié. Les résultats qui se situent dans les limites de tolérance habituelles (écart-type=± 0,13%) correspondent à des prises, d'essai de 1,5 à 2 mg.

     

Studies on the preparation and exchange reactions of 5-deuterated uracils

The mechanism of base catalyzed proton exchange at the 5-position of uracil and its N-methylated derivatives has been studied. These reactions proceed by addition — elimination across the 5,6-double bond when the 1-nitrogen is substituted with a methyl group, or with anchimeric assistance of the N-1 anion if the 1-position is unsubstituted. The base catalyzed hydrolyses of 1,3-dimethyluracil and 3-methyluracil also appear to proceed through hydrated intermediates. A facile method for an acid catalyzed preparation of 5-deuterated uracils is described as well as a simple and accurate method for analysis of deuterium content.