An Enzymatic Procedure for the Preparation and Purification of 3′-Phosphoadenosine 5′-Phospho- [35S]Sulfate ([35S]PAPS): Applications in Syntheses of 8-Azido and 8-Bromo Derivatives of [35S]PAPS (original) (raw)

1996, Analytical Biochemistry

This paper describes a rapid and an efficient procethe ''active sulfate'' used as a primary donor of sulfuryl dure for the enzymatic synthesis of 3-phosphoadenosgroups (0SO 3) in biological systems (1). Sulfotransferine 5-phospho[ 35 S]sulfate ([ 35 S]PAPS). [ 35 S]PAPS was ases are a family of enzymes that catalyze the transfer synthesized by incubating ATP and a carrier-free [ 35 S]of the sulfuryl group from PAPS to a variety of acceptor Na 2 35 SO 4 with ATP sulfurylase, a recombinant APS kisubstrates including xenobiotics, proteins, glycolipids, nase and inorganic pyrophosphatase. The transfer of steroids, and glycosaminoglycans (2-6). Since the dis-35 SO 4 group from [ 35 S]Na 2 SO 4 to [ 35 S]PAPS proceeded covery of active sulfate, numerous chemical and enzymore efficiently in the presence of an ATP-regeneratmatic attempts have been made to synthesize both ing system composed of pyruvate kinase and phosadenosine phosphosulfate (APS) and PAPS. In biologiphoenol pyruvate. About 90% of the radioactivity prescal systems, PAPS is synthesized through the coordient in the starting material [ 35 S]Na 2 SO 4 was transnated action of three enzymes as outlined below (7). ferred to [ 35 S]PAPS within a 2-h reaction incubation. The reaction products were applied to a Mono Q column, and [ 35 S]PAPS was eluted by a step-wise gradient ATP / SO 20 4 S ATP sulfurylase APS / PPi [I] of triethylamine bicarbonate buffer (pH 7.5). Under these conditions, [ 35 S]PAPS eluted as a sharp peak at APS / ATP S APS kinase PAPS / ADP [II] 0.7 M triethylammonium bicarbonate and it was very well separated from other contaminants. The purified PPi / H 2 O S Pyrophosphatase 2 Pi [III] [ 35 S]PAPS (yield 85%, purity ú95%) was functional in donating sulfate to an oligosaccharide acceptor in a The products of above reactions are metabolically unstandard sulfotransferase reaction. The enzymatic stable and are rapidly degraded to mixtures of free procedure described above was particularly useful for sulfate, PAP, APS, and AMP. Among these, APS and the synthesis of [ 35 S]PAPS at a wide range of concen-PAPS are also acid-labile compounds losing their sultrations and specific activities (up to 1500 Ci/mmol). fate substitutions under acidic conditions (7). Commer-This generally useful approach was also found to be cially available [ 35 S]PAPS is extremely costly and is successful in the syntheses of 8-azido and 8-bromo deoften not sufficiently pure for the enzymatic studies rivatives of [ 35 S]PAPS. Applications of these two derivatives of PAPS, for purification and identification of that require PAPS at a known concentration and spesulfotransferases, have also been discussed. ᭧ 1996 cific activity. A number of chemical methods have been Academic Press, Inc.