Ch. Choi et al., DETERMINATION OF MET-ENKEPHALIN IN PHEOCHROMOCYTOMA CELLS (PC12) USING NONISOTOPIC METHODS, Molecules and cells, 6(4), 1996, pp. 400-404
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.