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.
Choi
Suk-Jung
SJ
Department of Chemistry, Kangnung National University, Gangneung 210-702, Korea. sjchoi@kangnung.ac.kr
Hwang
Jung Min
JM
Kim
Sung Il
SI
D000818:Animals; D002175:Candida / Q000201:enzymology; D003124:Colorimetry; D049832:Decapodiformes / Q000201:enzymology; D004112:Dimercaprol / Q000031:analogs & derivatives* / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism*; D008049:Lipase / Q000378:metabolism*; D013997:Time Factors
D004112:Dimercaprol; C014496:2,3-dimercaptopropan-1-ol tributyrate; D004228:Dithionitrobenzoic Acid; D008049:Lipase