Fluorometric Assay for Alcohol Sulfotransferase & PAPS Synthetase through Regeneration of PAPS

• Main Authors: Wei-Ti Chen, 陳偉迪
• Other Authors: Yuh-Shyong Yang
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/29334420779002328864

碩士 === 國立交通大學 === 生物科技系所 === 93 === The sulfoconjugation of biomolecules occurred widely in biological system which resulted in a dramatic change in the physicochemical property of the sulfonated compounds. Previously, 3’-phosphoadensine 5’-phosphosulfate synthetase (PAPSS) and sulfotransferases (STs) were found to involve in the course of sulfonation. The former activates inorganic sulfate to form PAPS, and the latter catalyzes the sulfuryl group of PAPS transferring to an acceptor molecule. The thesis is divided into three chapters, and introduces sensitive fluorometric assays developed for investigation into their fundamental issues concerning the role of sulfonation, such as the effects of quaternary structure on sulfotransferase function. First chapter described the convenient expression and purification procedure of recombinant STs by affinity column. Furthermore, a first continuous fluorometric assay was developed for alcohol sulfotransferase (AST). It utilized PAPS regenerated from 3’-phosphoadenosine 5’-phosphate (PAP) by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate (MUS) as the sulfuryl group donor. The change of fluorescence intensity of 4-methylumbelliferone (MU) corresponded directly to the amount of active AST and was sensitive enough to measure ng or picomole amount of the enzyme activity. Some properties of hDHEA-ST were determined by this method and were found comparable to published data. In second chapter, this coupled-enzyme assay was applied to study some effects of quaternary structure on AST. Some catalytic properties and thermal stability were compared between dimeric (wild-type) and monmeric (mutant) cytosolic STs. Final chapter described molecular cloning, expression, and purification of recombinant PAPSS. By coupling with the physiological reaction of PST, the activities of recombinant PAPSS were also determined fluorometrically. The two fluorometric methods developed not only are useful for the routine and detailed kinetic study of these important class of enzymes but also have the potential for the development of a high-throughput procedure using microplate reader.