2.

Materials

2.1. Preparation of Actived Solid Phases

2.1.1. Preparation of Thiol-Agarose and Titration of Thiol Groups

1. Sepharose 4B (Pharmacia BTH, Uppsala, Sweden)

2. Epichlorohydrin (1-chloro-2,3-epoxypropane) (Sigma).
3. Sodium thiosulfate (Fluka AG, Buchs, Switzerland).
4. Dithiothreitol (DTT) (Sigma).
5. 2,2’-Dipyridyldisulfide (2-PDS) (Sigma).
2.1.2. Preparation of 2-Pyridyldisulfide Agarose and titration of Pyridyl Disulfide Groups
1. Mercapto pyridine
2. 2,2’-Dipyridyldisulfide (2-PDS)
3. DTT
2.1.3. Preparation of Thiolsulfonate-Agarose (TS-gel)
1. Perhydrol (30% Hydrogen peroxidase)
2.1.4. Preparation of Thiolsulfinate-Agarose (TSI-gel)
1. Magnesium monoperoxyphthalate (MMPP)
2. Potassium ferricyanide
2.1.5. Titration of Thiol-Reactive Structures in the Agarose Derivatives
1. Reduced glutathione
2. 2,2’-Dipyridyldisulfide (2-DPS)
2.2. Immobilization of E. coli β-Galactosidase Onto Thiol-Reactive Agarose
For each of the following techniques it is necessary to have:

1. Empty PD-10 columns.

2. End-over-end mixer-
3. Vacuum pump.

2.2.1. Activity Determination

1. Activity buffer: 0.1 M potassium phosphate buffer, pH 7.5, containing 3 mM

magnesium chloride-

2. o-Nitrophenyl-β-D-galactopyranoside (ONPG)

2.2.2. Immobilization of the Enzyme

1. Thiolsulfonate-agarose, prpared as described in subheading

3. Methods

3.1. Preparation of Activated Solid Phases

3.1.1. Preparation of Thiol-Agarose

The preparation of mercaptohydroxypropil ether agarose gel (thiol-agarose) is carried out

essentially as described by Axén et al. 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 M NaOH.

2. Add slowly eith 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.

3.1.2. Thiol Group Analysis

1. The thiol content of both soluble and insoluble material is determined

spectrophotometrically by titration with 2,2’-dipyridyldisulfide (saturated solution,
1.5 mM, prepared by 30 min aditation followed by filtration) dissolved in 0.1 M
sodium phosphate, pH 8.0, according to Brocklehurst et. al.

3.1.2.1. Preparation of 2-pyridyldisulfide-Agarose

The preparation of 2-pyridyldisulfide-agarose is carried out as reported by Oscarsson et al.

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.

3.1.2.2. Titration of Pyridyldisulfide Groups

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 10 3 M-1
cm-1.

1. A filter-dried gel aliquot is weighed and suspend 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 Thiosulfonate-Agarose (TS-Gel)

1. Suction-dried thiol-agarose (15 g containing 500-800 µmoles 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 M sodium acetate, pH 5.0, at 4°C until use.
5. The thiolsulfonate group contet of the gel thus obtained is between 250 to 400
µmoles TS groups per g of dried gel.

3.1.4. Preparation of Thiolsulfinate-Agarose (TSI-Gel) in Two Steps

3.1.4.1. 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 M NaCl, 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.
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 M sodium phosphate buffer, pH 6.3, and suspend it in
15 mL of the same buffer.
6. Add 15 mL 2 M sodium 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 M sodium bicarbonate
buffer, pH 8.5.
9. Dissolve 3 g DTT in 15 mL 1mM ethylene 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 M sodium bicarbonate buffer, pH 8.5.
b. Distilled water.
c. 0.1 M acetic acid, until absence of DTT.

These conditions will give a thiol-agarose derivate containing between 400 and 600
µmoles of thiol groups per g dried gel (see Note 5).

3.1.4.2. Thiosulfinate-Agarose (TSI-Gel)

1. Suspend 15 g of suction-dried S2-Gel in 100 mL 0.2 M sodium acetate, pH 5.0, in

which the required amount of magnesium monoperoxyphalate 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 M sodium 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 M sodium
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-µL aliquots of supernatants with 3.0 mL of 1.5 mM 2,2’-dipyridyldisulfide
dissolved in 0.1 M sodium phosphate buffer, pH 8.0.
6. Measure the absorbance at 343 nm.