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A continuous spectrophotometric assay for the determination of diamondback moth esterase activity
Author(s) -
He Xiaodun
Publication year - 2003
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.10103
Subject(s) - esterase , diamondback moth , hydrolysis , chromatography , substrate (aquarium) , azadirachtin , chemistry , enzyme , biology , biochemistry , plutella , lepidoptera genitalia , pesticide , botany , ecology , agronomy
Conventional methods to determine esterase activity from insects are composed of a three‐step process where the enzyme is allowed to hydrolyze a 1‐naphthyl acetate substrate, that reaction is quenched by a SDS detergent, and then a Fast Blue B dye complex is formed with 1‐naphthol, the product of 1‐naphthyl acetate hydrolysis. These methods measure dye‐product complex rather than the product, 1‐naphthol. A new assay is presented that continuously monitors the formation of 1‐naphthol with the hydrolysis of an esterase substrate. The esterase activity was determined as the slope of the linear regression change in absorbance over time at 320 nm. The continuous assay provides a simple, rapid, and sensitive method for measuring esterases extracted from a single diamondback moth in 1–10 min. The detection limit of the assay is approximately 0.6 μM 1‐naphthol. The 1‐naphthol product from the esterase reaction was confirmed by HPLC analysis. According to the assay, the K m and V max values of the esterase were 28 ± 2 μM and 6.0 ± 0.1 μM/min, respectively, at 37°C for 1‐naphthyl acetate. The K i value was 9 ± 2 μM using azadirachtin, an insecticide from neem tree, Azadirachta indica (A.Juss). Azadirachtin was a reversible competitive inhibitor of the esterase activity. Arch Insect Biochem Physiol 54:68–76, 2003. Published 2003 Wiley‐Liss, Inc.