See QUMAS Change Request number SOP-DEK-ENZ53.
To standardize a procedure for the enzymatic assay of α-glucosidase by the modified Boehenger procedure at Sigma-Aldrich St. Louis.
This procedure applies to all products that have a specification for α-glucosidase activity.
3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
3.2. Unit Definition – One unit will convert 1.0 micromole of maltose to 2 moles of D-glucose per minute at pH 6.0 at 25 ºC.
3.3. ATP – Adenosine-5’-Triphosphate
3.4. ATP – Adenosine-5’-Diphosphate
3.5. G-6-P – Glucose-6-Phosphate
3.6. NADP+ – β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized form
3.7. NADPH – β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced form
3.8. G-6-PDH – Glucose-6-Phosphate Dehydrogenase
Maltose + H2O α - Glucosidase > 2(α - D - Glucose)
2(α - D - Glucose) + 2(ATP) Hexokinase > 2(G - 6 - P) + 2(ADP)
2(G - 6 - P) + 2NADP+ G-6-PDH > 2(6 - Phosphogluconate) + 2(NADPH) + 2H+
It is the responsibility of all Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
Temperature = 25 °C, pH = 6.0, A340nm, Light path = 1 cm
Spectrophotometric Stop/Endpoint Determination
7.3.1 7.3.1. 100 mM Sodium Acetate, 0.05% (w/v) Ethylenediaminetetraacetic Acid, Tetrasodium, Tetrahydrate, pH 6.0 at 25 °C (Buffer)
Prepare a 13.61 mg/ml solution of Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625 and a 0.55 mg/ml solution of Ethylenediaminetetraacetic Acid, Tetrasodium, Tetrahydrate, Sigma-Aldrich Product Number Sigma-Aldrich Product Number ED4SS in purified water. Adjust pH to 6.0 at 25 °C.
7.3.2 20% (w/v) Maltose (Maltose)
Prepare a 200 mg/ml solution in purified water using Maltose, Monohydrate, Grade I, Sigma-Aldrich Product Number M5885.
7.3.3 Glucose HK Assay Reagent (HK)
Immediately before use, dissolve contents of one vial of Glucose (HK) Assay Reagent, Sigma-Aldrich Product Number G3293, per volume on label.
7.3.4 α-Glucosidase (Enzyme)
Immediately prior to pipetting into reaction mixture, prepare a solution containing 3-5 units/ml in cold purified water.
7.4 STEP 1 – ENZYME STOP REACTION
7.4.1 Start a boiling water bath and bring to a rolling boil.
7.4.2 Pipette (in milliliters) the following Reagents into suitable containers:
7.4.3. Mix by inversion and allow to equilibrate to 25 °C. Then add:
7.4.4. Mix by swirling and incubate in a 25 °C water bath for exactly 5 minutes. Immediately remove vials from the 25 °C water bath and place in the boiling water bath. After 5 minutes of boiling, add:
7.4.5. Boil for 5 additional minutes and then centrifuge for 5 minutes. Decant the supernatant for use in 7.5 (Step 2).
7.5. STEP 2 – ENZYME ACTIVITY DETERMINATION
7.5.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
7.5.2. Allow to equilibrate to 25 °C using a suitable thermostatted spectrophotometer and record the initial A340 nm (AI) of all test and blank reaction mixtures. Then add:
7.5.3. Mix by inversion and monitor the increase in A340 nm until the Δ A340nm / minute is ≤ 0.0020, and this rate is maintained for a minimum of five minutes. Record the final A340 nm (AF). The corrected A340nm should be in the range of 0.20 to 0.55.
7.6.1 ΔA = AF - AI
|7.6.2||Units/mg of enzyme =||
(ΔA Test - ΔA Blank)(3.00) (2.00) (df)
|(6.22)(2)(0.10)(5)(mgs of enzyme)|
7.6.3. 3.00 = Volume (in milliliters) of reaction mixture in 7.5 (Step 1)
2.00 = Volume (in milliliters) of reaction mixture in 7.4 (Step 2)
df = Dilution factor of enzyme
6.22 = Millimolar extinction coefficient of NADPH
2 = µmoles of NADPH per µmole of Maltose
0.10 = Volume of enzyme used in the reaction mixture of 7.4 (Step 1)
0.10 = Volume of enzyme used in the reaction mixture of 7.5 (Step 2)
5 = Incubation time in minutes
7.7 FINAL ASSAY CONTENTRATION:
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