Assay Development for Lysyl Hydroxylase

Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides and the corresponding hydroxylated peptide using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, mass spectrometry and CD spectroscopy. A new HPLC method for efficient separation of lysine- from hydroxylysine-containing peptides has been developed in both organic (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). Peptides derivatization is done prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine- containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.