Sulfotransferase Enzyme Activity Assay Method Development.

摘要

Sulfotransferases are enzymes that catalyze the transfer of sulfo groups from a donor, for example 3'-phosphoadenosine 5'-phosphosulfate, to an acceptor, for example the amino or hydroxyl groups of a small molecule, xenobiotic, carbohydrate, or peptide. These enzymes are important targets in the design of novel therapeutics for treatment of a variety of diseases. This thesis examines the determination of the optimal sequence to use heparin biosynthetic enzymes for preparing bioengineered heparin and to develop assays for this important class of enzyme, paying particular attention to sulfotransferases acting on carbohydrates and peptides and the major challenges associated with their analysis.;The optimal order to use the biosynthetic enzymes on their natural polysaccharide substrates was deduced so that the appropriate defined substrates could be determined for each enzyme assay. Based on this study, it appeared that the order of enzyme treatment did not have a profound effect on the structures of the product as determined through its disaccharide compositinoal analysis.;A new method has been developed to assess the sulfotransferase enzyme activity directly based on measuring the products of heparosan and N-sulfo (NS) heparosan defined substrates of various chain lengths using hydrophilic interaction liquid chromatography-Fourier transform mass spectrometry (HILIC-FTMS). This method is fast accurate, and does not require the workup usually necessary for mass spectrometric methods. This enzyme activity assay's viability has been demonstrated and activity for 2-OST and 6-OST-3 have been determined in units of pmol product formed˙min-1˙mg-1 protein. The activity and kinetic constant data of commercially purchased 6-OST-3 has also been compared to that of laboratory-prepared enzyme.;Another method to assess the sulfotransferase enzyme activity directly has been developed without the use of radiolabel based on measuring the products of 2-aminoacridone (AMAC) labeled heparosan and NS heparosan defined substrates of various chain lengths using capillary electrophoresis - laser induced fluorescence (CE-LIF). Utilizing an AMAC tag affords ease in the separation of substrate/products from the assay reaction mixture and thus highly specific measurements and accuracy for determining kinetic information about the enzyme. Efficient CE-LIF conditions for running the reaction substrate of AMAC-NS heparosan decasaccharide have been determined. NS hexasaccharide and NS decasaccharide have also been tested as substrates to determine the reaction conversion.