Abstract
The spectrophotometric assay of 2,4-dienoyl coenzyme A (CoA) reductase (EC 1.1.1.34) was modified to improve the linearity and sensitivity of this method. 5-Phenyl-2,4-pentadienoyl-CoA, which has an absorbance maximum at 340 nm with an extinction coefficient of 44,300 M−1 cm−1, was synthesized and used as substrate. This compound is reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 2,4-dienoyl-CoA reductase to 5-phenyl-3-pentenoyl-CoA. When a tissue homogenate serves as an enzyme source, the product is further metabolized by Δ3Δ2-enoyl-CoA isomerase (EC 5.3.3.8) to 5-phenyl-2-pentenoyl-CoA, which is hydrated to 5-phenyl-3-hydroxypentanoyl-CoA by enoyl-CoA hydratase (EC 4.2.1.17). The modified assay method, which measures the decrease in absorbance at 340 nm due to the reduction of 5-phenyl-2,4-pentadienoyl-CoA and the oxidation of NADPH, is linear for a longer period of time and is twice as sensitive as the conventional assay with 2,4-decadienoyl-CoA as substrate.
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Abbreviations
- A:
-
absorbance
- CoA:
-
coenzyme A
- HPLC:
-
high-performance liquid chromatography
- NADPH:
-
nicotinamide adenine dinucleotide phosphate, reduced form
- Pi :
-
phosphate
- U:
-
unit
- UV:
-
ultraviolet
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Nada, M.A., Shoukry, K. & Schulz, H. Spectrophotometric assay of 2,4-dienoyl coenzyme a reductase with 5-phenyl-2,4-pentadienoyl-coenzyme a as substrate. Lipids 29, 517–521 (1994). https://doi.org/10.1007/BF02578250
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DOI: https://doi.org/10.1007/BF02578250