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, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides 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.