A new method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests

Abstract

A new method has been developed to determine the extent of proline hydroxylation in collagen. This method relies on the measurement of changes in the ratio of [3H]:[14C]-proline in bacterial collagenase digests, resulting from specific loss of tritium from the 4-trans position of the proline during hydroxylation. Proline hydroxylation can be measured in the presence of large amounts of noncollagen proteins, and simultaneous quantitation of the rates of collagen and noncollagen protein synthesis is obtained. The dual-labeled proline method is simple, rapid, and accurate. There was a very good correlation between this method and both ion-exchange chromatography and chloramine-T oxidation/tolue..