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
Precautions
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.
1. α-Amylase Sample Assay
a. Pipette (in mL) the following reagents into suitable containers:
| Reagent | Sample 1 | Sample 2 | Sample 3 | Sample Blank |
| Starch solution | 1.00 | 1.00 | 1.00 | 1.00 |
b. Mix by swirling and equilibrate to 20 °C. Then add (in mL):
| Reagent | Sample 1 | Sample 2 | Sample 3 | Sample Blank |
| α-Amylase Sample | 0.50 | 0.70 | 1.00 | – |
c. Mix by swirling and incubate for exactly 3.0 minutes at 20 °C. Then add:
| Reagent | Sample 1 | Sample 2 | Sample 3 | Sample Blank |
| Color Reagent | 1.00 | 1.00 | 1.00 | 1.00 |
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):
| Reagent | Sample 1 | Sample 2 | Sample 3 | Sample Blank |
| α-Amylase Sample | 0.50 | 0.30 | – | 1.00 |
f. Cool solutions on ice to room temperature.
g. Then add (in mL):
| Reagent | Sample 1 | Sample 2 | Sample 3 | Sample Blank |
| Ultrapure water | 9.00 | 9.00 | 9.00 | 9.00 |
h. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Samples and Sample Blank.
2. Standard Curve Preparation
a. Prepare a standard curve by pipetting (in mL) the following reagents into suitable containers:
| Reagent | STD1 | STD2 | STD3 | STD4 | STD5 | STD6 | STD7 | STD BLK |
| 0.2% (w/v) Maltose Standard | 0.05 | 0.20 | 0.40 | 0.60 | 0.80 | 1.00 | 2.00 | – |
| Ultrapure water | 1.95 | 1.80 | 1.60 | 1.40 | 1.20 | 1.00 | – | 2.00 |
| Color Reagent | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
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):
| Reagent | STD1 | STD2 | STD3 | STD4 | STD5 | STD6 | STD7 | STD BLK |
| Ultrapure water | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 |
e. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Standards and Standard Blank.
Calculations
06/17-1
