Comprehensive analysis of collagen metabolism in vitro using [43H] [14C]proline dual-labeling and polyacrylamide gel electrophoresis

John F. Bateman, Vince Harley, Danny Chan, William G. Cole

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A method to simultaneously quantify the production, secretion, and prolyl hydroxylation of individual types of collagen in cell culture samples has been developed. Collagens were biosynthetically labeled with a mixture of [14C]proline and [4-3H]proline. The labeled collagens were isolated and their component α-chains were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Migration of the collagen α-chains was determined by fluorography, and radioactivity in excised bands was quantified by scintillation counting. [14C]Proline labeling of collagen chains was used to determine the production and secretion of the different types of collagen. The ratios of the component α1(I) and α2(I) chains of type I collagen were also determined in this way. Prolyl hydroxylation of collagen α-chains was readily determined by measurement of their 3H:14C ratios. Following 4-hydroxylation, 3H was lost from the [4-3H]proline with alteration of this ratio. This dual-labeling method is suitable for the comprehensive analysis of collagen metabolism in multiple samples.

Original languageEnglish
Pages (from-to)171-175
Number of pages5
JournalAnalytical Biochemistry
Issue number1
Publication statusPublished - 1 Jan 1988
Externally publishedYes


  • cell culture
  • collagen metabolism
  • electrophoresis
  • proline hydroxylation

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