Covalent binding of thiols to thiolsulphinate-containing supports

Thiolsulphinate groups have been introduced on to pearl-polymerized agarose, cross-linked dextran, microcrystalline cellulose, polyacrylamide-dextran co-polymers, co-polymerized (ethylene glycol)-methacrylate, acrylic beads and porous glass. The procedure consists of a thiolation and two oxidation reactions. Depending on the conditions and type of solid phase used, degrees of substitution ranging from 60 to 500 μmol of thiolsulphinate groups per g of dried support were obtained. These groups provide the solid phase with the property of reversible covalent binding of thiol compounds via disulphide-bond formation. The experimental conditions for the introduction of thiolsulphinate groups on to agarose have been optimized, as this is one of the most widely used supports in chromatography. With agarose, degrees of thiolsulphinate substitution ranging from 100 to 500 μmol/g of dried derivative can be easily prepared from the same thiol-agarose batch by varying the concentration of the reagent used in the second oxidation step. The thiolsulphinate-agarose had the ability to bind up to 500 μmol of low-M r thiols/g of dried gel. The maximum binding capacities for thiolated BSA and Bence-Jones protein were 276 mg/g of dried gel (4.2 μmol/g) and 411 mg/g of dried gel (16.4 μmol/g) respectively. The thiol-sulphinate-agarose derivatives, regardless of their original degree of substitution, showed no decrease in thiol-binding capacity when stored for 75 days at 4°C.