ACE Kit - WSTA502 Product Manual

 Angiotensin-converting enzyme (ACE) is one of the key elements responsible for vasopressor action. ACE converts angiotensin-I to angiotensin-ll, a potent vasopressor, in the rennin-angiotensin system and contributes to increasing blood pressure by inactivating bradykinin, a strong antihypertensive peptide. Recently, various functional foods have received attention because of their inhibitory activity toward ACE.

 ACE activity is conventionally determined by UV measurement of the hippuric acid produced from the synthetic substrate Hyppuryl-His-Leu. However, the assay process is complicated and requires organic solvent. In this kit, a safe and straightforward modified method has been developed. 

 The colorimetric detection system in the ACE Kit-WST determines the amount of 3-hydroxybutyric acid (3HB) generated from 3-hydroxybutyryl-Gly-Gly-Gly by ACE. The kit is designed for 96-well microplate assays and is suitable for multiple sample measurements. No organic solvent extraction is required. The ACE Kit - WST assay is safe, simple, and provides highly reproducible data. 

Figure 1  Principle of the ACE inhibition assay using ACE Kit - WST

	50 tests	100 tests
Substrate buffer	1 ml × 1	1 ml × 2
Enzyme A	× 1	× 2
Enzyme B	× 1	× 2
Enzyme C	× 1	× 2
Coenzyme	× 1	× 2
Indicator solution	5 ml × 1	5 ml × 2

 

Store the kit at 0-5 °C. The kit is stable for 6 months.

• Several kit components are in glass vials. Please handle with care.
• Multiple measurements (triplicates of each sample) are recommended to obtain accurate data.
• If the water solubility of the sample is low, use dimethylsulfoxide or ethanol to dissolve. Then, dilute the solution with an appropriate buffer. The final concentration of organic solvent should be <1%.
• If the sample solution is acidic, adjust the pH to ≥5 before use for measurement.
• Ascorbic acid may interfere with the assay. The concentration of ascorbic acid in the sample solution should be <0.01% w/v. If the sample solution contains insoluble materials, remove it by centrifugation or filtration before use for measurement.

- Microplate reader (450 nm filter)

• 96-well microplate
• 2-20 μl, 20-200 μl, and 100-1000 μl pipettes
• Incubator
• Multi-channel pipette
• Disposable syringes (1 ml)

Enzyme working solution:

Dissolve Enzyme B in 2 ml of deionized water to prepare Enzyme B solution. Then add 1.5 ml of Enzyme B solution to Enzyme A to prepare Enzyme working solution.

• Enzyme A and B vials are capped under vacuum pressure. Add deionized water or solution through the rubber septum with a syringe, and then remove the septum.
• The Enzyme working solution is stable at -20 °C for 2 weeks or in a refrigerator for 3 days.

 

Indicator working solution:

Dissolve Enzyme C and Coenzyme in 3 ml of deionized water each. Add 2.8 ml of Enzyme C solution and 2.8 ml of Coenzyme solution to Indicator solution to prepare Indicator working solution.

• Enzyme C and Coenzyme vials are capped under vacuum pressure. Add deionized water through the rubber septum with a syringe, and then remove the septum.
• The Indicator working solution is stable at -20 °C for 2 weeks or in a refrigerator for 3 days.

Confirmation of the presence or absence of ACE inhibition

Prepare 100 μl of each sample.

• If the sample volume is <100 μl, please dilute the sample.
• If the sample is colored, prepare 150 μl of each sample.

Colorless sample

See Table 1 and Figure 2

1. Add 20 μl of sample solution to each sample well.
2. Add 20 μl of deionized water to each blank 1 well and 40 μl to each blank 2 well.
3. Add 20 μl of Substrate buffer to each well.
4. Add 20 μl of Enzyme working solution to each sample well and blank 1 well.
   • Enzyme working solution is easy to remain on the well wall. Please tap the plate to ensure that all solutions have been mixed completely.
   • Since the enzymatic reaction starts immediately after adding the Enzyme working solution, use a multi-channel pipette to minimize the well-to-well time lag.
5. Incubate at 37 °C for 1 h.
6. Add 200 μl of Indicator working solution to each well.
7. Incubate at room temperature for 10 min.
8. Read the absorbance at 450 nm using a microplate reader.

 

Confirmation of the presence or absence of ACE inhibition

 

Colored sample

See Table 2 and Figure 4

1. Add 20 μl of sample solution to each sample well and sample blank well.
2. Add 20 μl of deionized water to each blank 1 well, 40 μl to each blank 2 well and 240 ul to each sample blank well.
3. Add 20 μl of Substrate buffer to each sample well, blank 1 well and blank 2 well.
4. Add 20 μl of Enzyme working solution to each sample well and blank 1 well.
   • Enzyme working solution is easy to remain on the well wall. Please tap the plate to ensure that all solutions have been mixed completely.
   • Since the enzymatic reaction starts immediately after adding the Enzyme working solution, use a multi-channel pipette to minimize the well-to-well time lag.
5. Incubate at 37 °C for 1 h.
6. Add 200 μl of Indicator working solution to each sample well, blank 1 well and blank 2 well.
7. Incubate at room temperature for 10 min.
8. Read the absorbance at 450 m using a microplate reader.

 

 

Confirmation of the presence or absence of ACE inhibition

Determination of IC₅₀

Dilute sample solution with deionized water.

      Dilutions: 1 (no dilution), 1/5, 1/5², 1/5³, 1/5⁴, 1/5⁵, 1/5⁶

Figure 6  Prepatation of sample solution

Colorless sample

See Table 3 and Figure 

1. Add 20 μl of sample solution to each sample well.
2. Add 20 μl of deionized water to each blank 1 well and 40 μl to each blank 2 well.
3. Add 20 μl of Substrate buffer to each well.
4. Add 20 μl of Enzyme working solution to each sample well and blank 1 well.
   • Enzyme working solution is easy to remain on the well wall. Please tap the plate to ensure that all solutions have been mixed completely.
   • Since the enzymatic reaction starts immediately after adding the Enzyme working solution, use a multi-channel pipette to minimize the well-to-well time lag.
5. Incubate at 37 °C for 1 h.
6. Add 200 μl of Indicator working solution to each well.
7. Incubate at room temperature for 10 min.
8. Read the absorbance at 450 nm using a microplate reader.
9. ACE inhibition can be calculated from the equation:
   ACE inhibition (%) = [(A_{blank 1} - A_sample)/(A_{blank 1} - A_{blank 2})] × 100

 

Determination of IC₅₀ (50% inhibitory concentration)

 

Colored sample

See Table 4 and Figure 9

1. Add 20 μl of sample solution to each sample well and sample blank well.
2. Add 20 μl of deionized water to each blank 1 well, 40 μl to each blank 2 well and 240 μl to each sample blank well.
3. Add 20 μl of Substrate buffer to each sample well, blank 1 well and blank 2 well.
4. Add 20 μl of Enzyme working solution to each sample well and blank 1 well.
   • Enzyme working solution is easy to remain on the wellywall. Please tap the plate to ensure that all solutions have been mixed completely.
   • Since the enzymatic reaction starts immediately after adding the Enzyme working solution, use a multi-channel pipette to minimize the well-to-well time lag.
5. Incubate at 37 °C for 1 h.
6. Add 200 μl of Indicator working solution to each sample well, blank 1 well and blank 2 well.
7. Incubate at room temperature for 10 min.
8. Read the absorbance at 450 nm using a microplate reader.
9. ACE inhibition can be calculated from the equation:
   ACE inhibition (%) = [{A_{blank 1} - (A_sample - A_{sample blank})}/(A_{blank 1} - A_{blank 2})] × 100

 

 

Determination of IC₅₀ (50% inhibitory concentration)

1. Le Hoang Lam, T. Shimamura, K. Sakaguchi, K. Noguchi, M. Ishiyama, Y. Fujimura and H. Ukeda, Anal. Biochem., 2007, 364, 104.
2. Le Hoang Lam, T. Shimamura, S. Manabe, M. Ishiyama and H. Ukeda, Anal. Sci., 2008, 24, 1057.