DETERMINATION OF MET-ENKEPHALIN IN PHEOCHROMOCYTOMA CELLS (PC12) USING NONISOTOPIC METHODS

Nonisotopic methods for the determination of biologically active subst ances have been introduced for safety and environmental reasons. In th e present study, reversed-phase high performance liquid chromatography (HPLC) and immunodot blotting methods as alternatives to overcome som e problems of radioisotopic methods were used to measure amounts of en kephalin in pheochromocytoma cells (PC12). The purification of enkepha lin from cell extract was carried out by using octadesylsilica minicol umn (Sep-Pak). Using HPLC, 4.16 ng of Met-enkephalin was detected in 1 0(7) PC12 cells but Leu-enkephalin was not. In order to detect a lower concentration of enkephalin, the immunodot blotting method was used. Spots were visualized using the avidin/biotin peroxidase complex (ABC) technique and nickel ammonium sulfate-glucose oxidase intensification (NASGOI) method. As the result of densitometry, the detection limit o f Met-enkephalin of the former method was approximately 3.13 ng and th at of the latter one about 0.39 ng. When using the NASGOI method, the cellular content of Met-enkephalin in 10(7) PC12 cells was 4.69 ng. Fr om the above results, it is thought that nonisotopic methods are to so me extent sensitive, reproducible, less costly, safer, and less labori ous than the isotopic methods for the determination of Met-enkephalin in PC12 cells.