sepharose and cyclen

Cysteine-containing biomolecules are attractive targets in the study of thiol biology. Here we introduce a novel method for the selective enrichment of thiol-containing molecules using a thiol-capture zinc(II) complex of 1,4,7,10-tetraazacyclododecane (Zn(2+)-cyclen). Recognition of N-acetylcysteine amide by Zn(2+)-cyclen has been studied by potentiometric pH titration, revealing formation of a 1:1 thiolate-bound Zn(2+)-cyclen complex with a large thiolate-affinity constant of 10(6.2)M(-1) at 25°C and I=0.10M (NaCl). The Zn(2+)-bound thiolate anion is unexpectedly stable in aqueous solution at pH 7.8 under atmospheric conditions for a few days. These findings have contributed to the development of a convenient method for separation of thiol compounds by using a micropipette tip. A 200μL micropipette tip containing 10μL of hydrophilic cross-linked agarose beads attached to Zn(2+)-cyclen moieties was prepared. All steps for thiol-affinity separation (binding, washing, and eluting) are conducted using aqueous buffers at room temperature. The entire separation protocol requires less than 15min per sample. We demonstrate practical example separations of cysteine-containing molecules. This micropipette tip method would be used preferentially as an alternative to existing tools for reliable enrichment of thiol-containing molecules.

Topics: Cyclams; Cysteine; Equipment and Supplies; Heterocyclic Compounds; Potentiometry; Sepharose; Zinc

A pyrene-labeled Zn²+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (∼300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn²+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.

Topics: Animals; Chelating Agents; Cricetinae; Cyclams; DNA; Fluorescent Dyes; Gels; HeLa Cells; Heterocyclic Compounds; Humans; Models, Molecular; Molecular Conformation; Pyrenes; Sepharose; Solubility; Staining and Labeling; Water; Zinc

4-(1,4,7,10-Tetraazacyclotetradec-1-yl)methylbenzoic acid (cycmba, 1) has been synthesized, as a step towards the eventual development of sequence-specific hydrolytic complexes. A cobalt(III) complex of 1, [Co(cycmba)Cl2]Cl.1.5H2O (.1.5H2O) was found to be active against both an activated phosphodiester compound, bis(nitrophenyl)phosphate (BNPP), and supercoiled DNA. The presence of the benzoate group depresses the rate of hydrolysis of the ligand-Co(III) system at neutral pH, as confirmed by the kinetics results of a methyl ester analog. The ability of (2.1.5H2O) to bind to solid substrates and remain active was also demonstrated by attachment of the molecule to agarose beads.

Topics: Carboxylic Acids; Catalysis; Chlorides; Cobalt; Cyclams; DNA; Heterocyclic Compounds; Hydrogen-Ion Concentration; Hydrolysis; Ligands; Microspheres; Molecular Structure; Potentiometry; Sepharose; Spectrum Analysis; Water