Development of a convenient peptide‐based assay for lysyl hydroxylase

Abstract

Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C‐5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2‐oxoglutarate; the latter is decarboxylated to form succinate and CO2. To develop a convenient, RP‐HPLC based LH assay, we used Fmoc solid‐phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP‐HPLC, MALDI‐TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP‐HPLC conditions. The best results were achieved using peptide derivatization (1‐anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP‐HPLC analysis. The products (di‐ and tetra‐substituted Lys‐ and Hyl‐containing peptides) were well resolved by RP‐HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP‐HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi‐step, require radio‐labeled substrates, and/or measure depletion of 2‐oxoglutarate or formation of CO2. Since the LH reaction with 2‐oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 330–338, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com