Isolation of a Specific Ascorbic Acid (Vitamin C) Oxidase

Abstract

An extract of Hubbard squash oxidizes both the synthetic and the natural ascorbic acid (vitamin C)∗ with great rapidity. This is due to an enzyme having an optimum pH of 5.83 to 5.96. It may be obtained and purified by extracting the squash (edible part) with twice its weight of 30% ethyl alcohol for 10 minutes. The centrifuged and filtered fluid is treated with an equal volume of acetone, which causes a yellow sticky substance to precipitate. This may be washed free of yellow pigment with acetone, dissolved in water and reprecipitated with acetone. A third precipitation yields a preparation, which after drying in vacuo over sulphuric acid has an activity 500 times that of the original extract.

This preparation is water soluble and gives slight protein tests. Alcohol and saturated solutions of neutral salts, however, do not precipitate it. It is digested (inactivated) by trypsin. A polysaccharide accompanies the enzyme in the above precipitation. We have found no way of removing it thus far.

This enzyme differs in various ways from the “hexoxidase” which v. Szent-Györgyi 1 discovered in cabbage leaves, e. g., the hexoxidase is precipitated by saturated (NH₄)₂SO₄ solution; it oxidizes not more than about 25% of the substrate, whereas the enzyme of the squash oxidizes 100% very rapidly. Moreover, the kinetics of our preparation point to the presence of a single enzyme. Substances thus far tested, such as cysteine, tyrosine, glutathione, and phenols, are not affected. We suggest, therefore, that the enzyme responsible be designated “ascorbic acid oxidase”.

It requires the presence of atmospheric oxygen, which plays the role of hydrogen acceptor. The oxidized ascorbic acid may be reduced to its original state by hydrogen sulphide. The enzyme is remarkably stable to dialysis, oxygen and carbon monoxid. Hydrogen sulphide, however, inactivates it.

For ascorbic acid estimation, Tillmans and associates 2 2,6-di-chlorobenzenoneindophenol method was employed.