On the Inactivation of Ascorbic Acid Oxidase*

Abstract

An outstanding characteristic of the enzymatic oxidation of ascorbic acid by highly purified preparations of ascorbic acid oxidase, is the pronounced inactivation of the enzyme that occurs during the course of the reaction (1). It is the purpose of this communication to present data showing that the enzyme inactivation occurring during the reaction, is due to causes which, for the purposes of discussion and clarification, may be divided into two categories: (1) Inactivation due to environmental conditions during the reaction; i.e., pH, substrate concentration, dilution, rate of shaking, etc., and (2) inactivation due to some factor inherent in the ascorbic acid-ascorbic acid oxidase-oxygen reaction. The first type of inactivation may be minimized by the use of the inert protein gelatin in the enzyme dilution under a prescribed set of environmental conditions (1). The second type of inactivation is not minimized to any great extent by the use of added gelatin. The groundwork for a study of the latter type of inactivation of ascorbic acid oxidase was laid by Steinman and Dawson (2), when they reported that catalase, peroxidase, and methemoglobin markedly protect ascorbic acid oxidase against inactivation. Inactivation encountered under various environmental conditions will be discussed first. Effect of Substrate Concentration on the Inactivation of Ascorbic Acid OxidaseWhen no inert protein is present in the reaction system, the enzymatic oxidation of ascorbic acid is characterized by a continuous decrease in the rate of oxygen uptake, until eventually the rate becomes zero, even though a considerable amount of unoxidized ascorbic acid may still remain in the flask. That this phenomenon is due to inactivation of the enzyme is evidenced by the fact that the addition of more enzyme to the system after the rate has approached zero results in a sharp increase in the rate of oxidation as measured by oxygen uptake. The total amount of oxygen absorbed by the system by the time the rate of oxidation approaches zero, will henceforth be called the "inactivation total" and will be expressed in mm. 3 oxygen absorbed. Table I shows how the inactivation total varies with the substrate concentration for a given amount of enzyme and how it is affected by the inert protein, gelatin.The data as given in columns 2 and 3 of this table show that when no gelatin is present in t.he reaction system, the amount of work (extent of oxidation) that a given amount of purified enzyme can accomplish before becoming completely inactivated decreases with increase in original concentration of ascorbic acid.

Reaction characteristics of the ascorbic acid oxidase of Myrothecium verrucaria

“…Dawson and co-worker's current interpretation of reaction inactivation (20) involves production of extremely low levels of peroxide that inactivate the enzyme which explains earlier observations related to protection by catalase. The same reports from Dawson's laboratory (20,101) suggest that some of the copper sites of the enzyme may function "non-enzymatically" in forming hydrogen peroxide but in such small quanti ties that the overall stoichiometry is not affected.…”

“…The same reports from Dawson's laboratory (20,101) suggest that some of the copper sites of the enzyme may function "non-enzymatically" in forming hydrogen peroxide but in such small quanti ties that the overall stoichiometry is not affected. Extend ing the study of the enzymatic copper, Poillon and Dawson (79) found twenty five percent in the Ou(I) state and seventy five percent in the Cu(II) condition.…”

“…Various literature reports (20,108) have demonstrated that hydrogen peroxide can be formed in the enzymatic or nonenzymatic oxidation of ascorbic acid. Typical data from these experiments are presented in Table 1.…”

Ascorbic acid oxidase of Myrothecium verrucaria

“…The purified ascorbic acid oxidase of higher plants also is susceptible to reaction inactivation and several studies of the mechanism have been reported (7,11,23). The mechanism with the atypical oxidase is probably different, however, since there is no evidence that sulfhydryl groups or copper are involved in catalysis.…”

Properties of the ascorbic acid oxidase of Myrothecium verrucaria in vivo

“…The work of Dawson and Tokuyama (11) indicates that the hydrogen peroxide, a secondary product, is produced by some of the copper sites on the enzyme which function non-enzymatically. The amounts of hydrogen peroxide decomposed by catalase would be too small to give a significant difference in measurements of oxygen uptake.…”