Immobilization of E coli fiGalactosidase Onto Thiol Reactive Agarose

3.2.1. Activity Determination

1. The free fi-galactosidase activity is determined using 14 mMONPG as a substrate in 0.1 M potassium phosphate buffer, pH 7.5, containing 3 mM magnesium chloride (activity buffer) (see Note 10). The released o-nitrophenol is determined spectrophotometrically at 405 nm (18).

2. The immobilized enzyme activity is assayed by incubating 100 |L aliquots of gel suspensions (containing 10 mg of suction-dried gel derivatives) with ONPG in activity buffer, using a 1 cm path length cuvet provided with magnetic stirring.

3.2.2. Protein Determination

1. The protein concentration is estimated from the absorbance values at 280 nm and using an extinction coefficient of 2.09 for a 1 mg/mL solution of E. coli fi-galac-tosidase (18).

2. The protein content of the gel derivatives is determined by total amino acid analysis, after extensive drying in a dessicator over phosphorus pentoxide and hydrolysis in 6 M HCl for 24 h at 110°C.

3.2.3. Preparation of Immobilized Enzyme Derivatives

1. Wash thiolsulfonate-agarose (TS-gel) in a sintered-glass filter connected to a vacuum pump, with 0.1 M potassium phosphate buffer, pH 7.0 (immobilization buffer) (see Note 11).

2. Transfer 2-g aliquots of suction-dried TS-gel equilibrated in immobilization buffer to empty PD-10 columns (see Note 12).

3. Add 7.5 mL of ß-galactosidase solutions (containing between 1.0 and 50.0 mg of protein, respectively) in 0.1 Mpotassium phosphate buffer, pH 7.0 (see Note 13).

4. Mix the suspensions gently in an end-over-end mixer for 16 h at 4°C.

5. Wash the resulting insoluble derivatives sequentially with 25-mL portions of 0.1 M potassium phosphate buffer, pH 7.0, with and without 0.5 M NaCl (see Note 14).

6. Equilibrate and dilute the conjugates to 22 mL with 0.1 Mpotassium phosphate buffer, pH 7.5, containing 3 mM magnesium chloride (activity buffer).

3.2.4. Blocking Excess Active Groups

1. Incubate for 30 min under mixing, 2.0 g of suction-dried gel derivatives with 20.0 mL of 8 mM glutathione solution. After incubation, filtrate, wash and dilute to 20.0 mL with activity buffer.

2. Assay ß-galactosidase activity in both the filtrates and blocked-gel suspensions (see Note 16).

3.2.5. Enzyme Elution

1. Incubate aliquots of suction-dried derivatives (100 mg) with 4.0 mL of fresh 50 mM DTT in 0.1 M sodium phosphate, pH 8.5, with mixing for 1 h under end-over-end rotation.

2. After filtration, determine activity in the filtrates (see Note 17).

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