[25] Preparation of ribose-1-phosphate

Publisher Summary Ribose-l-phosphate (R-l-P) is formed by the phosphorolytic cleavage described by Kalckar. The preparation reaction is catalyzed by nucleoside phosphorylase and pulled by oxidizing the hypoxanthine formed with xanthine oxidase. The reaction may be followed by determining the amount of acid-labile phosphate formed, using the Fiske–SubbaRow and Lowry–Lopez reactions. A rather unfractionated enzyme may prove satisfactory if care is taken that the preparation does not contain any phosphatase. For preparation of the barium salt of ribose-l-phosphate mixture of phosphate buffer, nucleoside phosphorylase, xanthine oxidase, and inosine is incubated at room temperature under an oxygen atmosphere and shaken vigorously. For preparation of cyclohexylamine salt fifty-four micromoles of barium R-1-P is dissolved in 0.225 ml. of water; 14.8 mg. of cyclohexylamine sulfate dissolved in 0.2 ml. of water is added. To the pooled supernatants 7 vol. of butanol is added, and the mixture is centrifuged to remove the amorphous precipitate. The crystals are collected by centrifugation, washed in ether, and dried. A purity of 99 % of the weight of the R-1-P cyclohexylamine salt could be accounted for as acid-labile phosphate in the Fiske–SubbaRow reaction.