Abstract
L-Ascorbic acid (AsA) is specifically required for the decarboxylation of α-ketoglutarate (KGA) in the prolyl 4-hydroxylase reaction, due to its structural characteristics. For further clarification of the role of AsA in proline hydroxylation, AsA and its oxidation products during proline hydroxylation were determined by high-performance liquid chromatography (HPLC) using a radioisotope analyzer as a detection system. AsA was oxidized in the uncoupled reaction system without a suitable peptide substrate or with an unsuitable one such as poly-L-proline. Furthermore, the oxidation of AsA occurred in the complete reaction, even in the presence of a high concentration of dithiothreitol (DTT) in the reaction solution. These results indicated that the uncoupled reaction inevitably occurred during proline hydroxylation and this uncoupled reaction was accompanied by the oxidation of AsA, which led to its consumption. The reaction pathway of this hydroxylation was also discussed.
