title: A novel thiol-affinity micropipette tip method using zinc(II)-cyclen-attached agarose beads for enrichment of cysteine-containing molecules.
abstract: 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.
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Hiroshi
authors: H
authors: Kusamoto
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Akio
authors: A
authors: Shiba
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Norinao
authors: N
authors: Koretake
authors: Laboratory of Organic Medicinal Chemistry, Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-0292, Japan.
authors: Haruto
authors: H
authors: Fujioka
authors: Laboratory of Organic Medicinal Chemistry, Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-0292, Japan.
authors: Yuhzo
authors: Y
authors: Hieda
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Emiko
authors: E
authors: Kinoshita-Kikuta
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan. Electronic address: kinoeiji@hiroshima-u.ac.jp.
authors: Eiji
authors: E
authors: Kinoshita
authors: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan. Electronic address: tkoike@hiroshima-u.ac.jp.
authors: Tohru
authors: T
authors: Koike
mesh_terms: D000080027:Cyclams; D003545:Cysteine / Q000737:chemistry*; D004864:Equipment and Supplies; D006571:Heterocyclic Compounds / Q000737:chemistry*; D011199:Potentiometry; D012685:Sepharose / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
chemical_list: D000080027:Cyclams; D006571:Heterocyclic Compounds; D012685:Sepharose; C038072:cyclen; D015032:Zinc; D003545:Cysteine
keywords: Macrocyclic tetraamine; Thiol-affinity chromatography; Thiolate recognition; Zinc(II) complex