A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

1991-11

AUTHORS

Francisco Batista-Viera, Mariela Barbieri, Karen Ovsejevi, Carmen Manta, Jan Carlsson

ABSTRACT

A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase. Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized. The gel bead can be regenerated and reused at least twice. Besides agarose, cellulose, crosslinked dextran, and polyacrylamide were shown to be very suitable as supports for solid-phase thiolsulfonates. More... »

PAGES

175-195

Journal

TITLE

Applied Biochemistry and Biotechnology

ISSUE

2

VOLUME

31

Author Affiliations

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  • Identifiers

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    http://scigraph.springernature.com/pub.10.1007/bf02921788

    DOI

    http://dx.doi.org/10.1007/bf02921788

    DIMENSIONS

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