24] Enzymic synthesis, purification, and fractionation of (2′-5′)-oligoadenylic acid

Abstract

Publisher Summary This chapter discusses the optimal synthesis of 2-5A. Methods to optimize the yield of 2-5A or to obtain radioactive material of known specific activity are described with the 2-5A synthetases from three sources containing relatively large amounts of the enzyme: interferon-treated mouse L or human HeLa ceils and rabbit reticulocytes. The chapter described methods for the purification and fractionation of 2-5A on a preparative scale (10-50 rag). The synthesis of 2-5A with the synthetase bound to a solid support has been more satisfactory than incubations in solution giving markedly higher yields of 2-5A per unit of extract, reflecting the removal (in large part) of degradative enzymes and the consequent ability to maintain the accumulation of the 2-5A products over a period of 6-12 days. There is, in addition, the obvious advantage of the use of the partially purified column- or paper-bound enzyme free of any unlabeled nucleotide pool, both with respect to the purity of product and the synthesis of radioactive 2-5A. The use of an ATP regenerating system based on creatine phosphate and creatine kinase identical to that used in many cell-free protein synthesizing systems has frequently resulted in slightly increased yields of 2-5A.