This procedure may be used for the determination of α-Amylase activity.
The spectrophotometric stop reaction determination (A540, Light path = 1 cm) is based on the following reaction:
Unit Definition: One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C.
Sodium phosphate, monobasic, Product Number S0751
Sodium chloride, Product Number S9888
Starch from potato, Product Number S2004
Sodium hydroxide, Product Number S5881
Potassium sodium tartrate, tetrahydrate, Product Number S2377
3,5-Dinitrosalicylic acid, Product Number D0550
D‑(+)‑Maltose, monohydrate, Product Number M9171
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.
Buffer solution (20 mM Sodium Phosphate with 6.7 mM Sodium Chloride, pH 6.9 at 20 °C) – Prepare a solution containing 2.4 mg/mL of sodium phosphate, monobasic, Product Number S0751, and 0.39 mg/mL of sodium chloride, Product Number S9888, in ultrapure water. Adjust to pH 6.9 at 20 °C using 1 M NaOH/1 M HCl.
Starch solution [1.0% (w/v) Soluble Starch Solution] – Prepare a 10 mg/mL solution using starch from potato, Product Number S2004, in Buffer:
2 M Sodium Hydroxide (NaOH) solution – Prepare a 80 mg/mL solution using sodium hydroxide, Product Number S5881, in ultrapure water.
5.3 M potassium sodium tartrate, tetrahydrate solution – Prepare a 1,496 mg/mL solution of potassium sodium tartrate, tetrahydrate, Product Number S2377, in 2 M Sodium Hydroxide (NaOH) solution. Dissolve solids by heating on a heating/stir plate with mixing. Do not heat to a boil!
96 mM 3,5-Dinitrosalicylic acid solution – Prepare a 21.9 mg/mL solution using 3,5‑Dinitrosalicylic acid, Product Number D0550, in ultrapure water. Dissolve solids by heating on a heating/stir plate with mixing. Do not heat to a boil!
Color Reagent solution – For preparation of 40 mL, add:
This solution is stable for 6 months at ambient temperature if protected from light. Volume prepared may be adjusted if needed.
0.2% (w/v) Maltose Standard – Prepare a 2 mg/mL solution in a volumetric flask using D‑(+)‑maltose, monohydrate, Product Number M9171, in ultrapure water.
α-Amylase Sample solution – Immediately before use, prepare a solution containing 0.75‑1.5 units/mL of α-Amylase in 20 °C ultrapure water.
Final assay concentration – In a 2.00 mL reaction volume, the final concentration is 0.50% (w/v) starch and ~1 unit of α-amylase.
a. Pipette (in mL) the following reagents into suitable containers:
b. Mix by swirling and equilibrate to 20 °C. Then add (in mL):
c. Mix by swirling and incubate for exactly 3.0 minutes at 20 °C. Then add:
d. Cover containers with a vented cap and place in a boiling water bath for exactly 15 minutes.
e. Remove containers from boiling water bath. Then add (in mL):
f. Cool solutions on ice to room temperature.
g. Then add (in mL):
h. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Samples and Sample Blank.
a. Prepare a standard curve by pipetting (in mL) the following reagents into suitable containers:
Note: Additional standard levels may be added as needed.
b. Cover containers with a vented cap and place in a boiling water bath for exactly 15 minutes.
c. Remove containers from boiling water bath. Cool solutions on ice to room temperature.
d. Then add (in mL):
e. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Standards and Standard Blank.
Calculations
where:
df = dilution factor
mL enzyme = mL of Sample added in step 1b.
6.
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