A study of collagen metabolism in cell cultures by fluorometric determination of proline and hydroxyproline

A technique of derivatization of proline (Pro) and 4-hydroxyproline (Hyp) by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole permitted the measurement of Pro and Hyp radioactivities, concentrations, and specific activities in the main fractions separated from cultures of fibroblast cells (extracellular collagen and non-collagen proteins, intracellular free Pro and Hyp, Pro- and Hyp-containing peptides, procollagen, and non-collagen proteins). The evaluation of collagen in the medium was obtained from as few as 10(4) cells. The method might advantageously replace [14C] Pro or [3H] Pro incorporation studies. It permits measurement of the size of the Pro pool and the amount of peptides formed by intracellular catabolism of collagen. It demonstrates that the time necessary for a full equilibration of intracellular Pro and intracellular collagen is longer than is generally believed. It avoids the uncertainties of protein labelling, which may vary with uncontrolled variations of the intracellular Pro specific activity.