Pyrroloquinoline Quinone → L-Ascorbic Acid

Participants

Reaction Steps

A reaction mixture consisting of 1.0 mM PMS, 25 mM potassium phosphate buffer (pH 7.0), 1.0 μM pyrroloquinoline quinone (PQQ), 1.0 mM CaCl2, 50 mM L-sorbosone and enzyme solution in a final volume of 100 μl with water is prepared just before the assay. The reaction is carried out at 30° C. for 60 min unless otherwise stated. The amount of vitamin C produced is measured at a wavelength of 264 nm by a high performance liquid chromatography (HPLC) which is coupled with a UV detector (TOSOH UV8000; TOSOH Co., Kyobashi 3-2-4, Chuo-ku, Tokyo, Japan), a dualpump (TOSOH CCPE; TOSOH Co.), an integrator (Shimadzu C-R6A; Shimadzu Co., Kuwahara-cho 1, Nishinokyo, Chukyo-ku, Kyoto, Japan) and a column (YMC-Pack Polyamine-II, YMC, Inc., 3233 Burnt Mill Drive Wilimington, N.C. 28403, U.S.A.). The amount of 2-KGA produced is measured by HPLC. One unit of the enzyme activity is defined as the amount of the enzyme that produces 1 mg vitamin C or 2-KGA in 60 min in the reaction mixture.

Provenance & Evidence