Enzymatic Assay of α-Glucosidase by the Modified Boehenger Procedure (EC 3.2.1.20)

Document History

See QUMAS Change Request number SOP-DEK-ENZ53.

1. Objective

To standardize a procedure for the enzymatic assay of α-glucosidase by the modified Boehenger procedure at Sigma-Aldrich St. Louis.

2. Scope

This procedure applies to all products that have a specification for α-glucosidase activity.

3. Definitions

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

4. Discussion

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⁺

5. Responsibilities

It is the responsibility of all Analytical Services laboratory personnel to follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
Temperature = 25 °C, pH = 6.0, A_340nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop/Endpoint Determination

7.3 REAGENTS:

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:

	Test1	Test2	Test3	Blank
Reagent 7.3.1 (Buffer)	1.40	1.40	1.40	1.40
Reagent 7.3.2 (Maltose)	0.50	0.50	0.50	0.50

7.4.3. Mix by inversion and allow to equilibrate to 25 °C. Then add:

Reagent 7.3.4 (Enzyme)

0.05

0.07

0.10

----

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:

Reagent 7.3.4 (Enzyme)

0.05

0.03

------

0.10

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:

Reagent 7.3.3 (HK)

2.90

2.90

2.90

2.90

7.5.2. Allow to equilibrate to 25 °C using a suitable thermostatted spectrophotometer and record the initial A_{340 nm} (AI) of all test and blank reaction mixtures. Then add:

Reagent 7.4.4 (Supernatant)

0.10

0.10

0.10

0.10

7.5.3. Mix by inversion and monitor the increase in A_{340 nm} until the Δ A_340nm / minute is ≤ 0.0020, and this rate is maintained for a minimum of five minutes. Record the final A_{340 nm} (A_F). The corrected A_340nm should be in the range of 0.20 to 0.55.

7.6 CALCULATIONS

7.6.1 ΔA = A_F - A_I

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:
NA

8. References & Attachments

NA

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required.