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.
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).
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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