The use of 7-chloro-4-nitrofurazan (NBD chloride) for the detection and evaluation of the hydroxyproline isomers

Abstract

This chapter describes assays for determining unlabeled or labeled trans-4-hydroxyproline and cis-4-hydroxyproline as well as two assays that can distinguish trans-3- from trans-4-hydroxyproline. 4-hydroxy-L-proline can be measured by colorimetric reaction that involves oxidation of hydroxyproline to pyrrole. This assay for hydroxyproline is applicable to the detection of hydroxyproline in a variety of connective tissues, cells in tissue culture, and purified collagens. Moreover, the radiochemical assay is based on the oxidation of hydroxyproline to pyrrole except that the hydroxyproline is converted to radioactive pyrrole, which can then be quantified using scintillation counting. An internal standard of unlabeled hydroxyproline is added to each sample prior to analysis, and its recovery is determined to correct the values obtained for the hydroxy[14C]proline. In addition, a rapid way to analyze radioactively labeled 3- and 4-hydroxyproline in a sample without resorting to an amino acid analyzer is the method of separation using high-voltage electrophoresis on paper.