A Colorimetric Microplate Assay Method for High Throughput Analysis of Lipase Activity

Abstract

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at 37 degrees C for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.

In VitroScreening of Medicinal Plants Used in Mexico as Antidiabetics with Glucosidase and Lipase Inhibitory Activities

“…Lipase inhibition assay was adapted from the method reported by Choi et al [13]. The assay is based on the spectrophotometric quantification of free thiols with chromogenic 5, 5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent), released by porcine pancreatic lipase from the 2,3-dimercapto-1-propanol tributyrate substrate (DMPTB, 97%).…”

Antioxidant property and α $$ \boldsymbol{\upalpha} $$ -glucosidase, α $$ \boldsymbol{\upalpha} $$ -amylase and lipase inhibiting activities of Flacourtia inermis fruits: characterization of malic acid as an inhibitor of the enzymes

“…The substrate p-nitrophenyl butyrate (PNPB) was dissolved in a 50 mM Tris-HCl buffer containing 6 % v/v Triton X-100 (pH 7.2) (Choi et al 2003). Candida rugosa lipase (Sigma L1754, 724 U/mg of protein) was dissolved in 10 mM TrisHCl buffer (pH 7.5, containing 10 mM KCl).…”

Purification and characterization of hepatic lipase from Todarodes pacificus

“…Major drawback of this method is the error caused by nonspecific reactions of 2,3-dimercapto-1-propanol tributyrate (DMPTB) and 5,5'-dithiobis (2-nitro benzoic acid) (DTNB) with various enzymes and chemicals present in tissue extracts (17). Although the problem of nonspecific reactions could be partially resolved by adding PMSF to the crude extract and by using a blank that did not contain DMPTB (17,18), the colorimetric assay method may not be sufficiently reliable when used for the discrimination of lipase from other esterases.…”

“…Lipase activity was routinely determined by the colorimetric assay method (18). Reaction mixtures containing 0.2 mM 2,3-dimercapto-1-propanol tributyrate (DMPTB), 0.8 mM 5,5'-dithiobis (2-nitro benzoic acid) (DTNB), 1 mM EDTA, 0.05 % Triton X-100, 50 mM Tris-Cl, pH 7.5 and enzyme were prepared in a 96-well microplate.…”