title: 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 Δ(1)-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.
authors: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan. Electronic address: irab@agr.ehime-u.ac.jp.
authors: Seiya
authors: S
authors: Watanabe
authors: Ehime Institute of Industrial Technology, 487-2 Kumekubota-machi, Matsuyama, Ehime 791-1101, Japan.
authors: Yoshinobu
authors: Y
authors: Hiraoka
authors: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.
authors: Shiori
authors: S
authors: Endo
authors: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.
authors: Yoshiaki
authors: Y
authors: Tanimoto
authors: Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors: Yuzuru
authors: Y
authors: Tozawa
authors: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.
authors: Yasuo
authors: Y
authors: Watanabe
mesh_terms: D000591:Amino Acid Isomerases / Q000378:metabolism; D000818:Animals; D003094:Collagen / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases / Q000378:metabolism; D006909:Hydroxyproline / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism
chemical_list: D003094:Collagen; D010088:Oxidoreductases; D006836:Hydro-Lyases; D000591:Amino Acid Isomerases; C021420:hydroxyproline epimerase; D006909:Hydroxyproline
keywords: Collagen; Enzymatic detection; L-hydroxyproline; trans-3-Hydroxy-L-proline; trans-4-Hydroxy-L-proline