Separation and evaluation of the cis and trans isomers of hydroxyprolines: Effect of hydrolysis on the epimerization

Abstract A procedure has been developed which can detect the hydroxyproline isomers trans -4-hydroxyproline (Hyp), trans -3-hydroxyproline, cis -4-hydroxyproline, and cis -3-hydroxyproline present in hydrolysates of collagens. The method involves hydrolyzing collagen, and reacting the primary amino acids with o -phthalaldehyde (OPA) and the hydroxyprolines and proline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) which combines specifically with secondary amino acids. The proline and hydroxyprolines are then separated by thin-layer chromatography and quantified by using a scanning spectrofluorometer. The method was used to show that both trans -4- l -hydroxyproline and trans -3- l -hydroxyproline were epimerized as a function of hydrolysis time to the cis isomers. An appreciable amount of trans -3-Hyp was degraded. Hydrolysis with 6 n HCl in the presence of 6% trichloroacetic acid gave greater epimerization than the 6 n HCl alone. Alkaline hydrolysis in 0.2 m Ba(OH) 2 caused more epimerization of trans -4-Hyp and trans -3-Hyp compared with acid hydrolysis but less degradation, so that alkaline hydrolysis is proposed for the evaluation of trans -3-Hyp, provided that the total of the cis and trans isomers be considered in this case.