Rapid determination of dabsylated hydroxyproline from cultured cells by reversed-phase high-performance liquid chromatography.
A high-performance liquid chromatographic method was modified for the determination of hydroxyproline in cultured cells derived from rat liver. First, the primary amino group in the cell hydrolysate was blocked with o-phthalaldehyde, then the secondary amino group was derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride). The dabsylated sample was treated with ethyl acetate to obtain a simple chromatographic elution profile of the cell hydrolysate. Dabsylhydroxyproline and proline were separated from other compounds by high-performance liquid chromatography in the gradient elution mode, and eluted at 4.71 and 8.00 min, respectively. The method was applied to the determination of hydroxyproline contained in cultured cells, the result being 25.4 +/- 3.6 pmol/microgram.
Sormiachi
K
K
Department of Microbiology, Dokkyo University School of Medicine, Tochigi, Japan.
Ikeda
M
M
Akimoto
K
K
Niwa
A
A
D000085:Acetates; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D006909:Hydroxyproline / Q000032:analysis* / Q000302:isolation & purification; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry / Q000166:cytology; D011392:Proline / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D009764:o-Phthalaldehyde; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000032:analysis
D000085:Acetates; D007202:Indicators and Reagents; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D009764:o-Phthalaldehyde; C007650:ethyl acetate; D011392:Proline; D004124:p-Dimethylaminoazobenzene; D006909:Hydroxyproline