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Assay of transglutaminase activity by electrophoretic removal of the unreacted monodansyl cadaverine
Authors:Eunhee Jeoung  Inyoung Ha  Suk-Jung Choi
Affiliation:1. Department of Chemistry, Gangneung-Wonju National University, 123 Jibyeondong, 210-702, Gangneung, Korea
2. AMOGREENTECH Co., Ltd., Gangneung Science Industry Park, 210-340, Gangneung, Korea
Abstract:Transglutaminases (TGases) catalyze the transfer of acyl groups between the γ-carboxyamide group of a glutamine residue and a primary amine. Rapid and precise determination of TGase activity is an important issue because improper function of TGases has been suggested to be associated with a variety of diseases. There have been tremendous efforts to develop the TGase assay methods to be more rapid, convenient and accurate. In the conventional assay method, fluorescence-tagged amine molecules such as monodansyl cadaverine (MDC) are coupled with casein by the action of transglutaminase. The removal step of unreacted MDC would require time-consuming work-up processes such as acid-precipitation and centrifugation. These processes would also interrupt the precise measurements of enzymatic activities. In this study, we have developed a new fluorometric assay methods to assay transglutaminase activity based on electrodialysis where the unreacted MDC is removed by electrophoresis. We have found the optimized condition to remove the unreacted MDC while preserving the β-casein protein. We also found the linear relationship between fluorescence intensity associated with β-casein and TGase can maintain in the range of 0–1.6 mU as well as 0–0.4 mU. The results show us as few as 0.1 mU of TGase could be detected by this method.
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