Abstract
A new method for the reversible immobilization of thiol-containing substances on agarose beads is presented. It is based on the use of thiolsulfinate (disulfide monoxide) as a solid-phase reactive group. The thiolsulfinate groups are introduced by controlled oxidation of thiol agarose. The method comprises two steps: First, mild oxidation of the agarose thiol groups to disulfide structures with potassium ferricyanide. Second, the oxidation of the so-formed agarose disulfide groups to thiolsulfinate groups by use of a stoichiometric amount of the oxidizing agent magnesium monoperoxyphtalate.
The solid-phase thiolsulfinate groups react very easily with thiols, which, as a result of the reaction, will be bound to the agarose beads by disulfide bonds. The adsorbent derivative is very suitable for the reversible immobilization of low as well as high-mol-wt thiols as demonstrated with reduced glutathione, penicillamine, mercaptoethanesulfonic acid, thiolated bovine serum albumin,β-galactosidase, and ±1-antitrypsine. Since treatment of the agarose derivatives with an excess of low-mol-wt thiols (e.g., dithiothreitol) leads to release of the bound molecules and regeneration of the original thiol groups, the reactive thiolsulfinate groups can easily be regenerated by the mentioned two-step procedure. The cycle of oxidation, binding, reduction, and reoxidation can be performed several times while retaining thiol binding capacity.
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Batista-Viera, F., Manta, C. & Carlsson, J. Solid-phase thiolsulfinates for the reversible immobilization of thiols. Appl Biochem Biotechnol 44, 1–14 (1994). https://doi.org/10.1007/BF02921846
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DOI: https://doi.org/10.1007/BF02921846