Enzymatic preparation of [2,4,5,6,8-14C, 5'-3H]guanosine for use in biosynthetic studies of pterinoid and flavin compounds.
To aid in investigations on the biosynthesis of pterinoid compounds, a procedure was developed for the preparation of double-labeled guanosine with 3H in the ribose moiety and 14C in the guanine ring. Ribose, as ribose-1-phosphate, was removed from [2,8,5'-3H]adenosine in a reaction using purine nucleoside phosphorylase and was coupled to unlabeled guanine using the same enzyme. The procedure was accomplished in one tube with no purification of intermediates necessary. The final product, [5'-3H]guanosine, was purified by affinity chromatography using a boronate column. The overall yield of labeled guanosine is about 91%. HPLC analysis indicates the radiochemical purity at 98%. The purity of the guanosine makes it suitable for in vivo studies of flavins, pterins, and other guanosine-derived compounds. A doubly labeled preparation was achieved by the addition of [5'-3H]guanosine to 14C-labeled guanosine in which only the guanine ring is labeled. The latter was formed in a manner similar to the one above. In this case, the labeled ribose moiety was enzymatically removed from [U-14C]guanosine and replaced with unlabeled ribose.