02 Oxidative Stress

ACE Kit - WST

ACE Kit - WST

ACE Inhibitory Activity Assay

  • Colorimetric Microplate Assay Possible
  • Possible to Test Multiple Samples at One Time
  • Simple Protocol
  • No Harmful Organic Solvent Required
    • Product code
      A502  ACE Kit - WST
    Unit size --
    50 tests Please inquire distributors about price.
    100 tests Please inquire distributors about price.

    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)

    Component
    50 tests ・Substrate Buffer   
    ・Enzyme A 
    ・Enzyme B 
    ・Enzyme C
    ・Coenzyme     
    ・Indicator Solution
    1 ml x 2
    x 2
    x 2
    x 2
    x 2
    5 ml x 2
    100 tests

Description

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.

 

Technical info

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)

Required Equipment and Materials

Comparison to the Conventional Method by Ethyl Acetate

Example Assay Data

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 (pink) or lacto tripeptide (blue) .
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.

Checking the Presence of ACE Inhibition

ACE inhibitions is present in the sample if absorbance of the sample (Total sample AU – Blank 2 AU) is lower than Blank 1 (Total Blank 1 AU – Blank 2 AU).

For 50 tests unit size, 14 samples can be tested in triplicate

Protocol: Presence of ACE Inhibition

Determination of IC50 (50% inhibitory concentration)

Prepare an inhibit ion curve using the sample concentration for X axis and the ACE inhibitory activity for Y axis. A typical inhibition curve is shown in Fig. 4.
Determine the concentration of the sample solution that gives 50% ACE inhibitory activity as indicated in Fig. 4 .

For 50 tests unit size, 2 samples can be tested in triplicate

Protocol: Determination of IC50

References

Open References

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.
4. Hiromichi Nakamura, et al., Antihypertensive Effects of Continuous Oral Administration of Nattokinase and Its Fragments in Spontaneously Hypertensive Rats. Biol. Pharm. Bull. 2011; 34(11) 1696 E701.
5. C. C. Lau, N. Abdullah and A. S. Shuoib, Novel angiotensin I-converting enzyme inhibitory peptides derived from an edible mushroom,Pleurotus cystidiosus O.K. Miller identified by LC-MS/MS, BMC Complement. Altern. Med., 2013, 13, 313.
6. K. Yamaki, Screening Research Methods for α-glucosidase Inhibitors and Angiotensin-converting Enzyme Inhibitors in Fermented Soybean Products and Fermented Milk Products ,JARQ, 2014, 48, 41.
7. C.Lau, N. Abdullah, A. Shuib, and N.Aminudin, Novel angiotensin I-converting enzyme inhibitory peptides derived from edible mushroom Agaricus bisporus (J.E. Lange) Imbachi identified by LC-MS/MS, Food Chemistry, 2014, 148, 396-401.
8. R. Nakabayashi, Z. Yang, T. Nishizawa, T. Mori and K. Saito, Top-down Targeted Metabolomics Reveals a Sulfur-Containing Metabolite with Inhibitory Activity against Angiotensin-Converting Enzyme in Asparagus officinalis, J. Nat. Prod., 2015, 78(5), 1179.
9. M. Alauddin, H. Shirakawa, K. Hiwatashi, A. Shimakage, S. Takahashi, M. Shinbo and M. Komaia, Processed soymilk effectively ameliorates blood pressure elevation in spontaneously hypertensive rats,J. Funct. Foods., 2015, 14, 126.
10. L. Basirico, E. Catalani, P. Morera, S. Cattaneo, M. Stuknyte, U. Bernabucci, I.De Noni, and A. Nardone, Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium, Journal of Dairy Science, 2015, 98(11), 7595-7601.
11. T. Hagi, M. Kobayashi, and M. Nomura, Metabolome analysis of milk fermented by gamma-aminobutyric acid-producing Lactococcus lactis, Journal of Dairy Science, 2016, 99(2), 994-1001.

Handling and storage condition

Handling and storage condition
0-5°C
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Price

Product Classification

Product Classification

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