Choi Suk-Jung, Hwang Jung Min, Kim Sung Il
Department of Chemistry, Kangnung National University, Gangneung 210-702, Korea.
J Biochem Mol Biol. 2003 Jul 31;36(4):417-20. doi: 10.5483/bmbrep.2003.36.4.417.
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.
本研究描述了一种基于5,5'-二硫代双(2-硝基苯甲酸)(DTNB)与2,3-二巯基-1-丙醇三丁酸酯(DMPTB)水解产物之间反应的比色微孔板法测定脂肪酶活性。在微孔板孔中制备含有DTNB、DMPTB和脂肪酶的反应混合物，在37℃孵育30分钟后记录405nm处的吸光度。通过该方法在0.1 - 1U脂肪酶活性范围内获得了线性关系。还针对0.01 - 0.1U或1 - 10U的范围优化了反应条件。在检测粗组织提取物时，DTNB与非特异性还原剂的反应产生了主要误差来源。然而，通过使用不含DMPTB的空白样品校正了该误差。
