A colorimetric microplate assay method for high throughput analysis of lipase activity

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.