alpha-Amylase Inhibitor (TBI5637)
$75.00 – $300.00
SKU | Stock | SIZE (mg) | Price | Quantity | ||
---|---|---|---|---|---|---|
TBI5637-1MG | Yes | 1 MG | $75.00 | |||
TBI5637-5MG | Yes | 5 MG | $300.00 |
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Description
Product Details
Form: Freeze-dried powder
Solubility: Distilled water or dilute buffer
Stability: Store at -20° C (-4° F)
Activity: 2000 U/mg protein
Protein: 50%
Unit Definition
One unit amount of inhibitor which, after preincubation for 20 minutes at 25° C, pH 6.9 will reduce the activity of two units of human salivary alpha-amylase by 50% (one unit of alpha-amylase will liberate, from soluble starch, one micromole of maltose per minute at 25° C, pH 6.9).
Assay Method
The reducing groups liberated from starch hydrolysis by salivary alpha-amylase, reduce 3,5-dinitrosalicylic acid resulting in formation of a colored product which can be measured spectrophotometrically at 540 nm. If the alpha-amylase inhibitor is present in the assay system it will inhibit alpha-amylase activity causing a corresponding decrease in the absorbance at 540 nm.
Applications
A proteinaceous inhibitor of the digestive enzyme alpha-amylase (1,4-a-D-Glucan glucanohydrolase; EC 3.2.1.1) has been isolated and purified from kidney beans Phaseolus vulgaris. It is a glycoprotein and it specifically inhibits corresponding plant, bacterial and fungal enzymes. Maximum inhibitory activity, against alpha-amylase, occurs at 37° C and pH 5.5. The inhibitor has a molecular weight of 45,000-50,000. Similar alpha-amylase inhibitors also occur in wheat, rye and some other beans.
Reagents
- 0.02 M Sodium phosphate buffer, pH 6.9 containing 0.006 M sodium chloride.
- Dinitrosalicylic acid color reagent. Dissolve 1.0 g 3,5-dinitrosalicylic acid in 20 ml 2 M NaOH. Add slowly, 30.0 g sodium potassium tartrate tetrahydrate. Dilute to a final volume of 100 ml with distilled water. Store in a tightly sealed container and protected from CO2. Stable for 2 weeks.
- 1% Starch. Dissolve 1.0 g soluble starch in 100 ml 0.02 M sodium phosphate buffer, pH 6.9, containing 0.006 M NaCl. Bring to a gentle boil to dissolve. Cool and make volume up to 100 ml, with distilled water, if necessary. Incubate at 25° C for 5 minutes prior to assay.
- Alpha-amylase/alpha-amylase inhibitor mixture. Prepare this solution fresh. It contains human salivary alpha-amylase (1-2U), alpha-amylase inhibitor (0.5-1U), human serum albumin (1.5 mg), calcium chloride (1.5 mg) in 0.02 solution phosphate buffer, containing 0.006 M NaCl, pH 6.9. Total volume of the mixture is 1.5 ml. This mixture is preincubated for 20 minutes at 25° C prior to assay.
Procedure
- Adjust spectrophotometer to 540 nm and 25° C and water bath at 25° C.
- Pipette 0.5 ml of the preincubated enzyme-inhibitor mixture into a series of numbered test tubes. Include a blank tube containing 0.5 ml distilled water and one containing the preincubated alpha-amylase (control) but without the inhibitor (see Reagent 4).
- Incubate tubes at 25° C for 5 minutes to achieve temperature equilibration.
- At timed intervals, add 0.5 ml starch solution (at 25° C) and incubate exactly 3 minutes at 25° C.
- Exactly 3 minutes following the addition of starch, add 1 ml dinitrosalicylic acid reagent to each tube at timed intervals.
- Place all tubes in a boiling water bath for 5 min.
- Cool to room temp and add 10 ml distilled water to each tube. Mix well.
- Read absorbance at 540 nm after zeroing the spectrophotometer with the blank.
- Determine micromoles of maltose liberated from a maltose standard curve.
Additional information
SIZE (mg) | 1 MG, 5 MG |
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Datasheet:
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