Development of a convenient peptide-based assay for lysyl hydroxylase.
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 CO(2). 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 CO(2). 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.
Cudic
Mare
M
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431-0991, USA.
Patel
Deepak A
DA
Lauer-Fields
Janelle L
JL
Brew
Keith
K
Fields
Gregg B
GB
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006900:Hydroxylation; D007700:Kinetics; D013058:Mass Spectrometry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D010455:Peptides; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase