3.1.1. Preparation of Thiol-Agarose
The preparation of mercaptohydroxypropyl ether agarose gel (thiol-agarose) is carried out essentially as described by Axen et al. (9). In this method the agarose beads (Sepharose 4B) are first reacted with epichlorohydrin in an alkaline medium. The oxirane groups thus formed are then converted with sodium thiosulfate to gel-bound thiosulfate groups (Bunte-salt), which are finally reduced with DTT to thiol groups (see Fig. 4).
1. Suspend 15 g suction-dried Sepharose 4B in 15 mL of 1 MNaOH.
2. Add slowly with agitation 2.5 mL epichlorhoydrin (see Note 1).
3. Incubate overnight at 22°C under shaking (see Note 2).
4. Transfer the epoxy-activated gel to a sintered-glass filter and wash it with distilled water. Use it immediately.
5. Equilibrate the gel with 0.5 Msodium phosphate buffer, pH 6.3, and suspend it in 15 mL of the same buffer.
6. Add 15 mL 2 Msodium thiosulfate and incubate overnight under shaking at 22°C (minimum 6 h).
7. Transfer the Bunte-salt gel to a sintered-glass filter and wash it with water. Store it in distilled water at 4°C until use (very stable) (see Note 3).
8. Suspend 15 g suction-dried Bunte-salt gel with 15 mL 0.2 Msodium bicarbonate buffer, pH 8.5.
9. Dissolve 3 g DTT in 15 mL 1 mMethylene diamine tetraacetic acid (EDTA) and add it to the Bunte-salt gel suspension (see Note 4).
10. Incubate the mixture during 1 h under shaking at 22°C.
11. Transfer to a sintered-glass filter and wash with:
a. 0.2 Msodium bicarbonate buffer, pH 8.5.
b. Distilled water.
c. 0.1 Macetic acid, until absence of DTT.
These conditions will give a thiol-agarose derivative containing between 400 and 600 ^moles of thiol groups per g of dried gel (see Note 5).
1. The thiol content of both soluble and insoluble material is determined spectrophoto-metrically by titration with 2,2'-dipyridyldisulfide (saturated solution, 1.5 mM, prepared by 30 min agitation followed by filtration) dissolved in 0.1 M sodium phosphate, pH 8.0, according to Brocklehurst et al. (17).
126.96.36.199. Preparation of 2-Pyridyldisulfide-Agarose
The preparation of 2-pyridyldisulfide-agarose is carried out as reported by Oscarsson et al. (4).
1. Suspend 10 g of suction-dried thiol-agarose gel in 40 mL of 50 mM sodium phosphate buffer, pH 8.5.
2. Dissolve 1.8 g of 2-mercaptopyridine and 3.5 g of 2,2'-dipyridyldisulfide in 40 mL of 98% ethanol.
3. Add this solution to the gel suspension and stir for 15 h at room temperature.
4. Wash with ethanol and water on a glass filter.
The determination is based on the release of 2-thiopyridone after reduction of 2-pyridyldisulfide-gel. The extinction coefficient at 343 nm for 2-thiopyridone is 8.02 x 103 M-1 cm-1.
1. A filter-dried gel aliquot is weighed and suspended in 25 mM DTT in 0.1 M phosphate buffer, pH 8.0.
2. After incubation under shaking at room temperature for 30 min, the supernatant is filtered and the absorbance at 343 nm is measured.
3.1.3. Preparation of Thiolsulfonate-Agarose (TS-Gel) (5,6)
1. Suction-dried thiol-agarose (15 g containing 500-800 |imoles SH groups/g dried gel) is suspended in 45 mL of 0.2 M sodium acetate, pH 5.0.
2. Add aliquots of 30% hydrogen peroxide under continuous shaking, 1.8 mL initially and then 2.2 mL after 30, 90, and 150 min. The incubation is then continued to give a total reaction time of 30 h (see Note 6).
3. Transfer the oxidized gel to a sintered-glass filter and wash with 0.1 M acetic acid until free of hydrogen peroxide.
4. Store the activated gel in 0.2 Msodium acetate, pH 5.0, at 4°C until use.
5. The thiolsulfonate group content of the gel thus obtained is between 250 to 400 | moles TS groups per g of dried gel.
3.1.4. Preparation of Thiolsulfinate-Agarose (TSI-Gel) in Two Steps (7,8)
188.8.131.52. Disulfide-Agarose (S2-Gel)
1. Suspend fifteen grams of suction-dried thiol-agarose gel in 30 mL of 0.1 M sodium phosphate buffer, pH 7.0, and add 0.1 M potassium ferricyanide (by 0.5-mL aliquots under shaking until the yellow color persists for at least 30 min).
2. Then wash the gel thoroughly on a sintered-glass filter with buffer, 1 MNaCl, and 0.2 M sodium acetate buffer, pH 5.0. It can be assumed that the disulfide (S-S) group content of obtained S2-gel is 50% of the thiol content in starting thiol-agarose.
184.108.40.206. Thiolsulfinate-Agarose (TSI-Gel)
1. Suspend 15 g of suction-dried S2-gel in 100 mL 0.2 Msodium acetate, pH 5.0, in which the required amount of magnesium monoperoxyphthalate has been dissolved (0.5 moles per mol of S-S groups) (see Note 7). The suspension is incubated while shaking for 2 h at room temperature (22°C).
2. Then thoroughly wash the gel derivative on a sintered-glass filter with 50 mM sodium acetate buffer, pH 5.0, and 0.1 M acetic acid.
3. The washed gel is stored at 4°C as a suspension in 0.2 Msodium acetate buffer, pH 5.0 (see Notes 8 and 9).
3.1.5. Titration of Thiol-Reactive Structures in the Agarose Derivatives
This is performed by back titration of remaining GSH free in solution, after its incubation with thiol-reactive gels. A blank is performed for spontaneous oxidation of GSH.
1. Equilibrate 2.0 g suction-dried gel aliquots (TS- or TSI-gel) with 0.1 Msodium phosphate buffer, pH 7.0, in centrifuge tubes.
2. Adjust the amount in each tube to 3.0 g with the same phosphate buffer .
3. Add 3-mL aliquots of 15 mM glutathione dissolved in the same buffer to each tube while mixing (Vortex).
4. Incubate the suspensions for 30 min at 22°C , mix every 5 min, and then centrifuge.
5. Mix 50-|L aliquots of supernatants with 3.0 mL of 1.5 mM2,2'-dipyridyldisulfide dissolved in 0.1 M sodium phosphate buffer, pH 8.0.
6. Measure the absorbance at 343 nm.
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