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Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test

Abstract

The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcohols by back-titration of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses commercial reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2–3 h.

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Figure 1
Figure 2: Diagram of 96-well microplate A for placement of enzyme and epoxides solutions at different concentrations.
Figure 3: Diagram of 96-well MTP (microplate C) for placement of buffer, NaIO4, epoxides (S1–S24), enzyme and L-adrenaline.
Figure 4: Diagram for the placement of buffer NaIO4, tributyrin (T), ethylene glycol bisoctanoate (G) and enzymes in the 96-well microplate E.
Figure 5: Diagram for the placement of buffer NaIO4, vegetable oils and enzymes in the microtest tubes.
Figure 6: Diagram of 96-well MTP F.
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Correspondence to Jean-Louis Reymond.

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Fluxá, V., Wahler, D. & Reymond, JL. Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test. Nat Protoc 3, 1270–1277 (2008). https://doi.org/10.1038/nprot.2008.106

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