[7] Enzymatic Synthesis and Separation of Retinyl Phosphate Mannose and Dolichyl Phosphate Mannose by Anion-Exchange High-Performance Liquid Chromatography

This chapter describes the enzymatic synthesis and separation of retinyl phosphate (Ret-P) mannose and dolichyl phosphate (DoI-P) mannose by anion-exchange high-performance liquid chromatography. Retinyl phosphate is chemically synthesized by the phosphorylation of all-trans-retinol by bisditriethylamine phosphate. GDP-mannose (Sigma) or GDP-[U-¹⁴C] mannose is used as the mannose donor for the preparation of unlabeled or [¹⁴C]mannose-labeled Ret-P-Mannose. Unlabeled Ret-P-Mannose is prepared. Ret-P-Mannose is purified from the extract by high performance liquid chromatography (HPLC) on a Mono Q HR 5/5 column as described in detail in the chapter. The concentration of Ret-P-Man is determined spectrophotometrically at 325 nm in 99% methanol. [¹⁴C]Mannose-labeled Ret-P-Man is prepared similarly to that for the synthesis of unlabeled Ret-P-Man. [¹⁴C]Man is purified from the extract by HPLC on a Mono Q HR 5/5 column is also described in the chapter. Typically 30–40% of the added [¹⁴C]mannose is transferred from GDP -[¹⁴C]mannose to exogenous Ret-P under these incubation conditions. [¹⁴C]Mannose-labeled Dol-P-Man may be prepared using incubation conditions similar to those described for the preparation of [14C]mannose- labeled Ret-P-Man. The major difference among the incubations is the inclusion of Triton X-100 which is required for the solubilization of the polyisoprenoid Dol-P. HPLC system for the separation of Mannose, Dol-P-Man, Ret-P-Man, and Ret-P is based on observation that Dol-P-Man, Ret- P-Man, and Ret-P could be separated by anion exchange chromatography on DEAE-Sephacel columns, utilizing ammonium acetate in 99% methanol as the eluent. Mannose, Ret-P-Man, mannose phosphate, and GDP-mannose are separated based on their difference in anionic charge on the anion exchange Mono Q HR 5/5 column. Dol-P-Man is eluted in this system with approximately the same retention time as Ret-P-Man but in very poor yields due to poor solubility of Dol-P-Man in 70% methanol.