Glucosidase, Beta Assay

Method: Salicin is hydrolyzed by β-glucosidase to yield saligenen and β-D-glucose. The rate of formation of glucose is measured in a hexokinase/glucose-6-phosphate dehydrogenase system. One unit releases one micromole of glucose per minute at 37°C and pH 5.0 under the specified conditions.

Reagents

• 0.1 M Acetate buffer, pH 5.0
• 1% Salicin solution. Prepare by dissolving 1 gm salicin in 100 ml 0.1 M acetate buffer, pH 5.0. Incubate at 37°C for 6 - 8 minutes before using.
• Glucose reagent system: 0.1 M Tris⋅HCl buffer pH 7.6, containing hexokinase (Worthington Code HKQL) ≥ 1.5 units/ml, ATP: 0.77 μmol/ml, NAD: 0.91 μmol/ml, and glucose-6-phosphate dehydrogenase (Worthington Code ZFL) ≥ 1.9 units/ml

Enzyme

Dissolve at one mg/ml in reagent grade water. Immediately prior to use dilute to 0.1 - 0.05 mg per ml in reagent grade water.

Procedure

Pipette 1.0 ml of respective enzyme dilutions into a series of numbered test tubes. Include a blank with 1.0 ml reagent grade water. Incubate tubes at 37°C for 6 - 8 minutes to achieve temperature equilibrium. At timed intervals add 4 ml of the salicin solution. Mix well. Incubate each sample exactly 10 minutes then stop reaction at timed intervals by immediately placing each tube in a boiling water bath for at least 5 minutes. Cool in ice bath.

Pipette 3.0 ml Glucose reagent system into cuvettes.

Determine A₃₄₀ of each solution before adding blank or enzyme-salicin reaction mixture. Add 0.1 ml blank and record change in A₃₄₀. To other cuvettes add 0.1 ml of the heated enzyme-salicin reaction mixture and record change in A₃₄₀. Record A₃₄₀ until no further change occurs in 3 - 5 minutes. Read final A₃₄₀.

Calculation