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For Research Use Only Products
- Colorimetric microplate assay
- Simple protocol
- No organic solvent required
- High reproducibility
Application: Angiotensin-converting enzyme (ACE) activity detection.
Screening of ACE inhibitors
Contents of the Kit:
|Enzyme A ||:2 tubes || Enzyme B ||:2 tubes |
|Enzyme C ||:2 tubes || Coenzyme ||:2 tubes |
|Substrate buffer||:1 ml x 2 || Indicator solution ||:5 ml x 2 |
Storage Condition: 0-5°C
Shipping Condition: ambient temperature
Required Equipment and Materials
plate reader with 450 nm filter; 96-well culture plate, 2-20 μl, 20-200 μl, 100-1000 μl and multi-channel pipettes; 37ºC incubator, Disposable syringe (1 ml)
The kit is used for the determination of ACE inhibition activity. ACE works in the Renin-Angiotensin system, which is one of the mechanisms of blood pressure control, to convert Angiotensin I to the vasopressor Angiotensin II. This enzyme also contributes to elevated blood pressure due to its role in breaking down the antihypertensive peptide Bradykinin. In recent years, food and supplements containing ingredients that block ACE have received attention for their use in preventing high blood pressure. The conventional method of measuring ACE inhibition employs the synthetic substrate Hippuryl-His-Leu. Hippuric acid from the synthetic substrate is extracted with ethyl acetate, condensed, redissolved, and then read at an absorbance of 228 nm. This method is cumbersome and measurement is subjected to error due to residual ethyl acetate. ACE inhibition Assay Kit enzymatically detects 3-Hydroxybutyric acid (3HB), which is made from 3-Hydryoxybutyryl-Gly-Gly-Gly (3HB-GGG). Using a 96-well format, it is possible to test multiple samples at one time. In addition, there is no need to use harmful organic solvents, resulting in a safe, simple, and highly reproducible assay.
Fig. 1 Principle of the assay system to determine ACE activity or inhibition activity.
Preparation of solutions
Enzyme working solution
Add 2 ml of purified water to Enzyme B bottle to prepare an Enzyme B solution.a) Then, add 1.5 ml of Enzyme B solution to the Enzyme A bottle to prepare a Enzyme working solution.b)
a)Enzyme A and B are freeze-dried and closed with a rubber cap under vacuum pressure. The contents may fly out of the container if the rubber cap is removed. Add purified water or Enzyme B solutions using a syringe, and then open the bottle after dissolving the contents.
b)The Enzyme working solution is stable at -20ºC for 2 weeks. If store in a refrigerator, stable for 3 days.
Indicator working solution
Add 3 ml of purified water to each of the Enzyme C and Coenzyme bottles and dissolve.c) Then, add 2.8 ml each of Enzyme C and Coenzyme to the indicator solution to prepare an indicator working solution.d)
c)Enzyme C and Coenzyme are freeze-dried and closed with a rubber cap under vacuum pressure. The contents may fly out of the container if the rubber cap is removed. Add pure water or Enzyme B solution using a syringe, and then open the bottle after dissolving the contents.
d)The Indicator working solution is stable at -20ºC for 2 weeks. If store in a refrigerator, stable for 3 days.
1. L. H. Lam, et al., Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid. Anal Biochem. 2007;364:104-111.
2. L. H. Lam, et al., Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyrate with water-soluble tetrazolium salt. Anal Sci. 2008;24:1057-1060.
3. L. H. Lam, et al., Flow injection analysis of angiotensin I-converting enzyme inhibitory activity with enzymatic reactors. Talanta. 2009;79:1130-1134.
Dilute sample solution with purified water.
example: dilution ratio: 1(no dilution), 1/5, 1/52, 1/53, 1/54, 1/55, 1/56
Table 1 Solution and buffer volumes in each well
1. Add 20 μl of the Sample solution (sample) or purified water (blank 1, blank 2) to each well.
2. Add 20 μl of the Substrate buffer to each well.
3. Add 20 μl of purified water to the blank 2 well.
4. Add 20 μl of the Enzyme working solution to the wells containing Sample solution and blank 1.
*3-Hydroxybutyric acid (3HB) is produced immediately upon addition of enzyme working solution. To reduce time lag from well to well, use a multichannel pippette.
5. Incubate the plate at 37°C for 60 minutes.
6. Add 200 μl indicator working solution to each well.
7. Incubate for 10 minutes at room temperature.
8. Measure the absorbance of each well at 450 nm.
9. Use the following equation to calculate the ACE inhibition activity (percent inhibition).
ACE inhibitory activity (inhibition rate %) = [(Ablank 1 – Asample) / (Ablank 1 – Ablank 2)] x 100
Fig. 2 Inhibition curves prepared by Alacepril and Captopril.IC50 of Alacepril and Captopril are 3.62 μM and 2.14 nM,
respectively. Both compounds are ACE inhibitors.
Fig. 3 Inhibition curves prepared by two beverages containing a
valyltyrosine (●) or lacto tripeptide (●).
IC50 of these beverages are 0.56% and 0.69%,
respectively. It is known that these substances have antihypertensive effects.
*Concentration of the beverage in the sample solution.
All Products are applicable only for life science research. Not for diagnostic research use.
Prices listed are for U.S. customer only and may vary in other countries.