Nonisotopic evaluation of collagen in fibroblasts cultures.
A method for the evaluation of collagen concentrations in the medium of fibroblasts in culture was developed. Collagen was precipitated with other proteins by addition of ethanol and hydrolyzed by 6 M HCl. The primary amino acids of the hydrolyzate were reacted with o-phthalaldehyde (OPA) and secondary amino acids (Pro, Hyp) were derivatized with 9-fluorenylmethyl-chloroformate (FMOC-Cl). The mixture was separated by isocratic HPLC on a reverse-phase column. FMOC-derivatives were detected by fluorometry, whereas OPA-derivatives were not. This method is suitable for the monitoring of collagen metabolism in fibroblast cultures exposed to various effectors.
Monboisse
V
V
Laboratory of Biochemistry, CNRS UA 610, UFR Medicine, Reims, France.
Monboisse
J C
JC
Borel
J P
JP
Randoux
A
A
D009327:4-Chloro-7-nitrobenzofurazan; D001205:Ascorbic Acid; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism*; D005449:Fluorenes; D006801:Humans; D006909:Hydroxyproline / Q000032:analysis; D011392:Proline / Q000032:analysis; D012867:Skin / Q000032:analysis; D009764:o-Phthalaldehyde
D005449:Fluorenes; D009764:o-Phthalaldehyde; D003094:Collagen; D011392:Proline; C054007:1-(9-fluorenyl)methyl chloroformate; D009327:4-Chloro-7-nitrobenzofurazan; D001205:Ascorbic Acid; D006909:Hydroxyproline