ON THE INACTIVATION OF ASCORBIC ACID OXIDASE*

Charles R. Dawson and Keiko Tokuyama

Department of Chemistry, Columbia University, New York, N.Y.

The enzyme ascorbic acid oxidase (AAO) is a blue, copper-containing protein

that catalyzes the aerobic oxidation of L-ascorbic acid to dehydroascorbic acid.
The reaction is readily followed manometrically, and when pure (catalase free)
AAO is employed the enzymatic oxidation is easily differentiated from the cu-
pric-ion-catalyzed reaction by the oxygen stoichiometry. The complete oxi-
dation of L-ascorbic acid as catalyzed by the enzyme requires the consumption
of 1 gram atom of oxygen, which is reduced to water. The oxidation when
catalyzed by free cupric ion, however, requires 1mole of oxygen (2 gram atoms)
per mole of ascorbic acid, and hydrogen peroxide is readily detected as a termi-
nal product (see FIGURE 1).
The blue color of the purified enzyme is one of its most characteristic prop-
erties. The intensity of the color per unit weight of copper is very much greater
than that of solutions of copper sulfate or of the copper-ammonia complex.’
The copper in the enzyme protein-copper complex is very tightly bound? It
is not removed by dialysis at physiological PH, nor by treatment with ion ex-
change resins? Furthermore, the copper of the resting (nonfunctioning) en-
zyme does not exchange with radioactive ( C U ~cupric
) ions when highly puri-
fied enzyme is employed.4g6 The activity of the enzyme is critically dependent
on its copper content, for when the copper is removed by dialysis against cyanide
the activity is simultaneously lost (FIGURE 2).
The properties of the purified enzyme are those of a globular protein having a
molecular weight of 150,000 and containing approximately 0.26 per cent copper,
corresponding to 6 copper atoms per molecule. Additional information about
the purified enzyme is summarized in TABLE 1.
One of the most characteristic properties of this copper oxidase is the marked
inactivation of the enzyme that occurs during the catalytic oxidation of the
substrate. The nature of the penomenon is indicated in FIGURE 3. The oxy-
gen-uptake curves shown in FIGURE 3 are typical of those obtained when a
purified sample of the enzyme is employed. When a relatively large amount
of the enzyme is used, a complete oxidation of the substrate occurs. However,
when a smaller amount of the enzyme is employed, the rate of the oxidation
reaction rapidly and continuously decreases until the enzyme is completely
inactivated. The “reaction inactivation” of the enzyme is indicated by an
“inactivation total,’’ that is, an incomplete oxidation of the ascorbic acid as
measured by oxygen uptake. The reaction inactivation of the enzyme is ir-
reversible, and further oxidation of the residual ascorbic acid does not occur un-
less more enzyme is added.
A number of years ago it was found in our laboratory that inert protein, such
as gelatin, protected the enzyme against inactivation; and certain agents, such
as native catalase and peroxidase, native or denatured methemoglobin, and
*The investigation reported in this a er was su ported in part by Grant A-3200(C1) from
the National Institute of Arthritis ana&etabolic &seases, Public Health Service, Bethesda,
Md.
212
 ENZYME-CATALYZE0 OXIDATION OF L- ASCORBIC ACID

RC R
HO-$' \o
+ 2 I0 2 + H20
HO-C, / AA0
CH
HO-CH HO-CH
CH~OH CH20H
I

CUPRIC ION -CATALYZED OXIDATION OF L- ASCORBIC ACID

8 F!
C
HO-C' \ 0-y/c\ 0
0
1'
W-C, / + O2 C" ++ 0-c /
'FH
+ H2°2

HGqH
FH HO-s: H
CH20H CH$H
I ~ X J R1.E ?'he aerolic oxidation of ascorbic acid as catalyzed by AAO and by free cupric

I
I
I
I
I
I
Enzyme activity I

e gin dialysis
vs. NQCN

Copper content

0 S 10 IS 20
Days
I ; I C : ~ ~ R E 2. Showing the stability of the enzyme's copper content and activity to dialysis
at physiological pH. Horizontal scale gives the days of dialysis. Note that both the ac-
tivity and copper content are rapidly lost when cyanide is added to the system.
213
2 14 Annals New York Academy of Sciences
hematin, were strikingly effective in protecting the enzyme against reaction
inactivation? In other words, these agents were found to increase markedly
the inactivation total per unit of enzyme. These studies in 1944 also indicated
that environmental factors such as the rate of shaking of the manometers, the

TABLE1
ASCORBICAcm OXIDASE'
Source: summer crookneck squash (Czcrczcrbita pep0 condensa)
Preparation: differential ultracentrifugation and fractional precipitation by
dialysis
Specific activity: 2000 U./mg.
Color: blue-green (dependent on 0%and substrate)
Homogeneity: 100 er cent (ultracentrifuge and electrophoresis)
Molecular weight: fSO,000
Copper: 0.26 per cent, corresponding to 6 copper atoms per mole
Activity per copper atom: 740 U.
Nitrogen: 16.8 per cent; no P or Ca

&b
3
Addexcess enzyme

0 20 30

FIGURE3. Showing how reaction inactivation becomes evident when relatively smaller
amounts of the enzyme are used.

PH of the system, the substrate concentration, and the enzyme concentration

were minor factors in the reaction inactivation. Because earlier experiments
in our laboratory8had shown that hydrogen peroxide was not a terminal product
of the ascorbic acid-AAO reaction, and the main product dehydroascorbic acid
 Dawson & Tokuyama: Ascorbic Acid Oxidase 21 5
was not harmful to the enzyme, it was suggested7 that the reaction inactivation
might be due to some factor inherent in the ascorbic acid-AAO-oxygen system,
possibly a highly reactive "redox form" of oxygen other than hydrogen peroxide
or H20.
The experimental work in 1944 also demonstrated that the inactivation total,
for any given amount of enzyme, decreased as the substrate concentration was
increased, but this inhibitory effect of the substrate was not further explored a t
that time.

-
10-

8-
A

=i
l80
o0l l4
A
._
C - 3
E
:6- <
01

c
60
0 a
1
2
-
->-
7
4- 0
0
"
I

2- I ;,I 20
40
I

I I I 0
3 2 I 0 10 20 30
- log [SI Time (min)
FIGURE4A. Showing how the apparent initial reaction velocity varies with the substrate
concentration, expressed as -log ( S ) .
B. Showing how the apparent initial reaction velocity and the reaction course vary with
increasing substrate concentration: curve 1: 4 x 10- M ascorbic acid; curve 2: 2 X lo-' M
ascorbic acid; curve 3: 1 X 10" M ascorbic acid; curve 4: 4 X 10-' M ascorbic acid.
Oxygen uptake was measured manometrically a t 25" C., pH 5.8. Each vessel contained
AAO (0.4gg.), 4 X lo-* M citrate-phosphate huffer, ascorbic acid, and distilled water to make
the final reaction volume 2.5 ml.
The substrate concentrations involved in FIGURE 4B are shown as dotted lines in FIGURE
4A.

During the past year we have carefully re-examined the possibility that sub-
strate inhibition might be an important factor in the reaction inactivation of
ascorbic acid oxidase. Our work has revealed that the reaction inactivation
can be differentiated from substrate inhibition. The major factor responsible
for the loss in activity of the enzyme during its catalytic function appears to be
an alteration of the enzyme by very small amounts of hydrogen peroxide pro-
duced by a secondary reaction during the enzymatic oxidation of the ascorbic
acid. Some of the experimental evidence in support of this conclusion may now
be reviewed.
The curves in FIGURE 4 show how the activity of the enzymes decreases (FIG-
URE 4A) and the inactivation of the enzyme increases (FIGURE 4B) as the sub-
216 Annals New York Academy of Sciences
strate concentration is increased beyond the optimum. It is apparent from
FIGURE 4 that substrate inhibition significantly increases the reaction inactiva-
tion; this is shown even more strikingly by the curves of FIGURE 5. In the
experiment corresponding to curve 1, the total substrate was added a t zero
time; it was an amount of substrate that resulted in a substrate concentration
slightly higher than the optimum. When the experiment was repeated under
the same conditions of enzyme concentration (except that the same total
amount of substrate was added stepwise, as indicated, in six equal increments
so that the substrate concentration was always below the optimum), curve 2
was the result. The enzyme accomplished much more oxidation before it be-
came completely inactivated, which was after the sixth substrate addition.
Although it is apparent from FIGURES 4 and 5 that the effect of substrate in-
hibition on the reaction inactivation is pronounced, it is also clear from curve 2
of FIGURE 5 that the inactivation is readily apparent even in the absence of
substrate inhibition. The loss in enzyme activity during the reaction must
therefore be due to some other factor or factors.
The experimental results indicated by the curves of FIGURE 6 establish that
the reaction inactivation cannot be ascribed to the products of the enzymatic
reaction or to any intermediates or secondary substances with concentration at
any given time dependent on the initial enzymatic reaction rate. The step-
wise addition of the substrate in the experiment corresponding to curve 2 re-
sulted in a lower rate of oxidation, as expected, but the rate and extent of en-
zyme inactivation was not correspondingly decreased; rather it was increased.
Consequently it may be concluded that the inactivation cannot be due to any
factor with a concentration dependent on the rate of the enzymatic oxidation.
Much new information about the nature of the inactivation phenomenon and
its cause was revealed when the enzyme was added incrementally to a given
amount of substrate (FIGURE 7).
In experiments 1 and 2 of FIGURE 7 the same quantities of ascorbic acid were
used. The total amounts of enzyme used were also the same; in experiment 1,
however, the total enzyme was added initially, whereas in experiment 2 the
enzyme was added at the periods indicated in increments of one fifth the amount
used in experiment 1. Only in the experiment involving the increment addi-
tions (curve 2) was enzyme inactivation apparent. Furthermore the apparent
enzyme inactivation was about the same for the third, fourth, and fifth enzyme
increments. In the case of the first enzyme increment the rate of the oxidation
was significantly lower than with the later increments. This was undoubtedly
the result of substrate inhibition, as discussed earlier.
In the experiment corresponding to curve 3, the amount of substrate was one
half that involved in experiments 1 and 2. The same quantity of enzyme,
however, was used as in each increment addition in experiment 2. It is to be
noted that the time required for enzyme inactivation was very similar to that
required for each enzyme increment in experiment 2. However, it is evident
from curve 3 that the enzyme accomplished much more oxidation before it be-
came inactivated. This point is particularly apparent if curve 3 is compared
with the 02-uptake curve resulting after the fifth enzyme increment addition in
experiment 2, where at the time of addition (75 min.) the amount of residual
 100

-- I 2 O l
801

..

Time (min.)
FIGURE5. Showing how the stepwise addition of concentrated substrate increases the apparent activity of the enzyme.
Oxygen uptake was measured manometrically at 25" C., pH 5.8. Each vessel contained AAO (0.4 pg.) 4 X M citrate-phosphate buffer,
6 X M ascorbic acid and distilled water to make the final reaction volume 2.5 ml. I n the experiment corresponding to curve 1, the total sub-
strate was added at zero time, while in experiment 2, the substrate was added in increments (total/6) as indicated.
218 Annals New York Academy of Sciences
unoxidized substrate was approximately one half the initial substrate concen-
tration (comparable to that used in experiment 3).
The lower rate and extent of oxidatipn resulting after the fifth enzyme in-
crement in experiment 2 (as compared with experiment 3) would suggest the
accumulation of an effective inactivating agent (such as H202) during the
previous 75 min. of the stepwise oxidation procedure used in experiment 2.

Theoretical
--- - 0, uptake-----

0 10 20 30 40
Time (min.)
FIGURE6. Showing how the stepwise addition of diluted substrate decreases the apparent
activity of the enzyme.
Oxygen u take was measured manometricall at 25" C., pH 5.8. Each vessel contained
AAO (0.4 p g f 4 X l(P M citrate-phosphate b d r , 2.4 X 1WgM ascorbic acid, and distilled
water to make the final volume 2.5 ml. I n the experiment corresponding to curve 1, the
total substrate was added at zero time, whilein the experiment 2, the substrate was added in
increments (total/3) as indicated.

In the case of experiment 2, a sixth increment of the enzyme was added at the
95-min. period; it was found that the extent of oxidation accomplished by the
enzyme and the time required for its inactivation was less than the previous
increments. Such results would be anticipated for a system gradually accu-
mulating an enzyme-inactivating agent such as H202 .
To check the possibility that simple autoxidation of the substrate might be
producing sufficient H202to account for the enzyme inactivation, the effect of
exposing the substrate to the reaction conditions in the absence of enzyme for
 ,,,Ir Theoretical O2 uptake

I
120-
- -X,
2

T
100-
-
=L
1

a
%
c
80-
0. &+--------
2

..
b
220 Annals New York Academy of Sciences
varying periods of time before initiating the enzymatic oxidation (by adding the
enzyme) was investigated. It was found through such pre-incubation exper-
iments that the initial reaction velocities, inactivation totals, and inactivation
times were not significantly different. I n other words, it could be concluded
that simple autoxidation of the substrate in the absence of enzyme could not
account for the reaction inactivation phenomenon.
In view of the evidence that a gradually accumulating by-product in the re-

I00

-- Catalase
-7 80
f
Y
0)
o
'a
3
60
* 0.
'0.
..-.--.
0-

40

20

0 I I
0 10 20 30 40 50 60 70 80
Time (min.)
FIGURE8. Showing the effects of adding catalase and gelatin a t various times on the
reaction course.
Oxygen values were measured manometrically at 25' C., pH 5.8. I n addition to the com-
ponents shown below, each vessel contained AAO (0.4 pg.), 4 X 1Oa M citrate-phosphate
buffer, 4 X 10-3 M ascorbic acid and copper-free water to make the final reaction volume 2.5
ml.
Experiment 1: no catalase or gelatin initially. 18pg. catalase was added later as indicated;
experiment 2: 18pg. catalase or 1 mg. gelatin was added to the buffered enzyme system before
initiating the reaction by addition of substrate; experiment 3: 18 pg. catalase wm added dur.
ing the reaction as indicated. Experiment 4: 1 mg. gelatin was added during the reaction as
indicated.

action, such as H202,might be responsible for the enzyme inactivation, i t

seemed advisable to reinvestigate carefully the effects of adding catalase and
gelatin (separately) before, during, and after the enzymatic reaction. The re-
sults are shown in FIGURE 8. Curve 1 (the control) shows that under the con-
ditions of the experiment, the enzyme in the absence of protective agents was
inactivated when only about 70 per cent of the ascorbic acid had been oxidized.
Complete oxidation of the ascorbic acid was rapidly obtained when a very
small amount of catalase, or a considerably larger amount of gelatin, was added
initially to the otherwise identical enzyme-ascorbic acid system (curve 2).
When the experiment was repeated (except that the same amount of catalase
 Dawson & Tokuyama: Ascorbic Acid Oxidase 22 1
was added after the oxidation had progressed for about 12 min.), a slight lag in
oxygen uptake occurred (curve 3); then the rate became essentially linear (no
further inactivation) until the ascorbic acid was completely oxidized. How-
ever, when gelatin was added during the course of the reaction (curve 4) there
was only a slight protective effect. I t should be noted that in the control ex-
periment (curve 1) catalase was added a t the 62-min. period after the enzyme
had been completely inactivated and a small evolution of gas had resulted
(presumably due to hydrogen peroxide decomposition). The addition of gela-
tin a t this point was without effect. Furthermore, the addition of catalase
after the rapid and complete oxidation of ascorbic acid by an excessive amount
of ascorbic acid oxidase never resulted in any evidence of hydrogen peroxide
accumulation.
The experimental results depicted in FIGURE 8 establish that the effectiveness
of catalase as a protective agent against the reaction inactivation of AAO is
very much greater than that of gelatin. The nature of the protective effect, as
well as the gas evolution observed when the catalase was added after the re-
action had proceeded for a period, are evidence that the catalase effect is the
result of its destruction of small amounts of hydrogen peroxide. The hydrogen
peroxide production cannot be a result of the main enzymatic reaction, for if
such were the case the reaction inactivation would be expected to vary directly
with the rate of the oxidation (FIGURE 6). It therefore appears that the inacti-
vation is due to very small amounts of hydrogen peroxide resulting from a
slower and secondary reaction. The gelatin protective effect can also be ra-
tionalized in terms of hydrogen peraxide production. It is known that the ef-
fect is observed with other proteins; it seems likely, therefore, that the gelatin
simply reacts chemically with the hydrogen peroxide in a rather slow and inef-
fective manner. When the gelatin is added initially it can cope with the slow
production of hydrogen peroxide and thereby protect the enzyme if a relatively
large amount (in comparison to the enzyme protein) is present. However,
when gelatin is added to the system after a certain amount of excess hydrogen
peroxide has accumulated, the gelatin cannot neutralize the hydrogen peroxide
rapidly enough to prevent effectively the continuing inactivation of the enzyme
(curve 4, FIGURE 8).
I t has been found that only very small amounts of hydrogen peroxide are
necessary to account for the observed enzyme inactivation. Furthermore.
additional experimental results have been obtained* that support the conclu-
sion that the hydrogen peroxide production is not a result of the main enzymatic
reaction but involves part of the protein-bound copper in a secondary way.
In other words, it is suggested that all of the copper in the enzyme may not be
simultaneously involved in the enzymatic activity.

Summary
During the aerobic oxidation of ascorbic acid catalyzed by the copper pro-
tein, ascorbic acid oxidase, the enzyme is rapidly and extensively inactivated.
The phenomenon has been reinvestigated, and new evidence concerning the
* K. ‘I’okupama and C. R. Dawson. 1Y61. “On the Mechanism of the Reaction Inac-
tivation of Ascorbic Acid Oxidase.” To be published.
222 Annals New York Academy of Sciences
cause of the reaction inactivation has been obtained. It has been found that
the enzyme inactivation is dependent on the time during which a secondary
product of the reaction may accumulate. The main products of the enzymatic
oxidation, that is, dehydroascorbic acid and water, appear not to be involved
in the inactivation phenomenon. However, a secondary product, hydrogen
peroxide, develops in small amounts during the reaction as the result of non-
enzymatic copper protein catalysis. In other words, it appears that some of
the copper sites on the enzyme protein function nonenzymatically in the sense
that they produce the secondary product, hydrogen peroxide, at a low rate.
Hydrogen peroxide is a very effective inactivating agent against ascorbic acid
oxidase, and only very small amounts are required to account for the experi-
mentally observable enzyme inactivation.
Acknowledgment
We are indebted to Stanley Lewis for technical assistance.
References
1. DAWSON, C. R. 1960. The copper protein, ascorbic acid oxidase. Ann. N.Y. Acad.
Sci. 88(2): 353.
2. DAWSON, C. R. 1950. Copper Metabolism. W. D. McElroy & B. Glass, Eds. : 18.
Johns Hopkins Press. Baltimore, Md.
3. JOSEMW, M. & C. R. DAWSON.1951. The copper of ascorbic acid oxidase. Experi-
ments with an ion exchange resin. J. Biol. Chem. 191: 1.
4. JOSELOW, M. & C. R. DAWSON.1951. The copper of ascorbic acid oxidase. Exchange
studies with radioactive copper. J. Biol. Cbem. 191: 11.
5. MAGEE,R. J. 1954. The enzyme, ascorbic acid oxidase. Experiments with radioac-
tive copper. Dissertation (microfilm copies available). Columbia Univ. New York,
N.Y.
6. DUNN,F. J. & C. R. DAWSON.1951. On the nature of ascorbic acid oxidase. J. Biol.
Chem. 189: 485.
7. POWERS, W. H. & C. R. DAWSON.1944. On the inactivation of ascorbic acid oxidase.
J. Gen. Physiol. 27: 181.
8. STEI", H. G. & C. R. DAWSON.1942. On the mechanism of the ascorbic acid-
ascorbic acid oxidase reaction. The hydrogen peroxide question. J. Am. Chem. SOC.
64: 1212.