Biological synthesis of ascorbic acid: <scp>l</scp>-galactono-γ-lactone dehydrogenase

Abstract

The terminal step in the biosynthesis of L-ascorbic acid in peas has been show-n to be the oxidation of L-galactono-y-lactone by an enzyme present in mitochondria (Mapson, Isherwood & Chen, 1954). This enzyme has been solubilized from mitocondria obtained from freshly germinated peas, cabbage leaves and cauliflower florets. Since the enzyme preparations from the mitochondria of the cauliflower florets were found to be more active than those from either peas or cabbage, we have used this material in most of the work reported here. The present paper describes the preparation and partial purification of the enzyme, together with an account of some of its properties. EXPERIMENTAL Ghemical8 L-Galactono-y-lactone was prepared by the reduction of D-galacturonic acid by borohydride as follows. D-Galact- uronic acid (10 g.) was dissolved in 40 ml. of water and neutralized with NaOH to between pH 8-5 and 9 0. Boro- hydride was added gradually with stirring at room temper- ature. Samples were removed and acidified with acetic acid to remove excess of borohydride, and galacturonic acid was tested for by boiling with Fehling's solution. After reduction was complete, the solution was acidified with acetic acid to pH 5-0, a slight excess of barium acetate added, and the precipitate filtered off. Ethanol (2 vol.) was added to the solution and the precipitate was collected. After the precipitate had been washed twice with 60% (v/v) ethanol, barium was removed by Dowex 50 resin, and to the filtrate 1-2 drops of 3 N-HCl were added. The solution was concentrated to a syrup and dried in vacuo. The lactone was recrystallized from absolute ethanol. L-Gulono-, D-altrono-, D-gluCono-and D-mannono-y- lactone were prepared as described by Isherwood, Chen & Mapson (1954). Glucurono--