An enzymatic method to estimate the content of L-hydroxyproline

Abstract

Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Δ1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the β-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004–1mM and 0.05–1mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma.