THE HYDROLYSIS OF STARCH BY HYDROGEN PEROXIDE

AND FERROUS SULFATE*

BY W. R. BROWN
(From Ihe Laboratory of Biochemistry, University of Cincinnati, Cincinnati)

(Received for publication, November 21, 1935)

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In 1924 Mathews (1) expressed the view that all matter exists in
two forms, stable and reactive, due to the energy content of the
molecule. On this basis he postulated that enzymes are sub-
stances which by their presence facilitate the transfer of energy
from some external source to the substrate, thus raising its energy
content and making it labile. The work of Hill (2), Boyd (3),
and Sigal (4) from this laboratory has added evidence to support
this view.
Since oxygen is such a common source of energy for living
matter, it is quite possible that amylase is a part of an oxidation-
reduction system, or is activated by such a system. On this
hypothesis was based a series of experiments to ascertain just how
closely the action of an oxidation-reduction chain upon starch
parallels the action of amylase. Hydrogen peroxide and ferrous
sulfate (Fenton’s reagent) were chosen as a suitable oxidation-
reduction system for the basis of experimental study.
Gatin-Grueewska (5) and Gerber (6), using hydrogen peroxide
alone, and Durieux (7), using hydrogen peroxide and ferric
chloride, had shown that starch is broken down by means of these
substances with the formation of dextrins and reducing substances.
All three concluded that the action was analogous to that pro-
duced by diastase. Omori (8), using several heavy metals with
hydrogen peroxide upon starch, secured the hydrolysis, but
concluded that the action was quite different from that of enzymes.

* Part of a dissertation submitted to the Graduate School of the Univer-

sity of Cincinnati in partial fulfilment of the requirements for the degree
of Doctor of Philosophy, June, 1934.
417
418 Starch Hydrolysis by H,O, and FeS04

EXPERIMENTAL

The experiments were carried out according to the following

scheme: 10 cc. of a 1 per cent soluble starch (Kahlbaum) solution,
2 cc. of acetate buffer (pH 3.8), 10 cc. of 1 per cent hydrogen
peroxide (commercial 30 per cent diluted to 1 per cent), and
2 cc. of 0.01 M ferrous sulfate in a large test-tube were covered with
toluene and placed in a bath at 37”. At various intervals small
portions were removed and tested with dilute iodine and the optical
rotation was observed. At other intervals small aliquots were

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taken and the acidity and cupric reducing power by the Bertrand
method were noted.
As the reaction proceeded, the solution became water-clear and
greatly reduced in viscosity. There was considerable production of
cupric reducing substances (Table I) and a continuous decrease
in the optical rotation of the solution (Table II). The tube con-
taining 2 cc. of ferrous sulfate showed a decrease in optical rotation
of 15.3 per cent of the original rotation in 3 hours. The tube con-
taining 4 cc. of ferrous sulfate showed a decrease of 84.0 per cent
in the same time. 6 cc. caused a decrease of 84.8 per cent in the
24 hours, while 8 cc. effected a decrease of 82.7 per cent in 2 hours.
The color produced by addition of dilute iodine changed from
blue to red and finally to colorless as the reaction proceeded. The
rate of splitting of starch was directly proportional t.o the amount
of iron added (Table II).
In addition to ferrous sulfate, which has been shown to be very
active in the catalysis of the action of hydrogen peroxide on
starch, several other salts, which show some catalytic action, were
tested. Ferric salts are of the same order of efficiency as the
ferrous. Copper and manganese salts are greatly inferior to the
iron. Complex salts, ferro- and ferricyanides, have very little
effect, while manganese dioxide has no effect whatever. The
efficiency of these salts bears no relationship to their peroxidase
activity as measured by the benzidine reaction. Copper is by far
the most efficient as a peroxidase, ferrous and ferric iron being
considerably weaker. Manganese, which is as effective in the
catalysis of starch as copper, has practically no peroxidase action.
Tests were made upon the solution to ascertain if the action were
a true hydrolysis with the liberation of free glucose or maltose.
 W. R. Brown 419

After the achromic point with iodine had been reached, the solution
reduced alkaline copper sulfate in the cold. This indicated a much
stronger reducing agent than the simple sugars. The addition of

TABLE I
Production of Reducing Substances
Reaction mixture: 10 cc. of 1 per cent starch, 2 cc. of acetate buffer
(pH 3.8), 10 cc. of 1 per cent HzO,, 5 cc. of 0.01 M FeS04. Temperature 37”.
-
Time 0.05 N Iodine reaction
CU
KMnOa

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hrs. cc. mo.
Control, no HzOs, no FeSOa. 24 0.60 1.89 Blue
“ H202, no FeS04.. 24 6.65 20.94 Deep violet
Reaction mixture. . 24 54.30 171.05 Colorless
I‘ “ . . . .. . . . .. . .. . . 20 51.00 160.70 ‘I
‘I 6‘ . . . .. . . . .. . .. . . 18 52.90 166.60 ‘I

TABLE II
Change in Optical Rotation during Reaction
Reaction mixture: 10 cc. of 1 per cent starch, 2 cc. of acetate buffer
(pH 3.8), 10 cc. of 1 per cent hydrogen peroxide, 0.01 M FeSOb added as
indicated. Temperature 37”. Water added to make total volume of 30 cc.
-

Experiment No. Iodine reaction Angle of rotation 2

__
E
To
3 4
_- k
hrs. iegrees hrs. d egrees hrs. degrees

1 (No Hdh) 20 Blue 1.50 3 1.5020 1.49 - -

2 28 Red 1.45 20 1.4028 1.27 + -
min
3 30 Colorless 1.50 3 1.2720 0.30 + +
min.
4 15 Pink 1.45 15 1.14 2.5 0.24 + +
5 15 ‘I 1.45 15 0.99 2.5 0.23 + +
6 20 Colorless 1.45 15 0.91 2 0.26 + +
-

phenylhydrazine caused the formation of an orange amorphous

precipitate which turned brown on standing but could not be
induced to crystallize. Glucose or maltose as such were not
present. The solution was tested with fuchsin-sulfur dioxide
420 Starch Hydrolysis by H202 and FeS04

reagent and with sodium bisulfite for the presence of free aldehyde.
Both of these tests were strongly positive, indicating that the
simple sugars were further attacked with the liberation of a free
aldehyde group. Neither hydrogen peroxide nor iron interferes
with any of these tests. There was no change in these tests after
the removal of hydrogen peroxide by the use of manganese dioxide.
Added amounts of hydrogen peroxide or ferrous sulfate to these
tests, carried out upon known solutions, had no effect.
Concentration of the reaction mixture in vacua at 3040” gave a

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distinctly acid distillate which did not reduce alkaline copper and
did not restore the color to fuchsin-sulfur dioxide, but gave a strong
reduction of ammoniacal silver nitrate. This was concluded to
be probably formic acid. There was considerable acidity left in
the residual solution. The acidity and aldehyde tests are con-
siderably increased by the addition of glucose or maltose to the
reaction mixture.
A solution of glucose or maltose, when treated with hydrogen
peroxide and ferrous sulfate, increased in acidity and decreased in
optical rotation. Formic acid and free aldehydes are produced.
Phenylhydrazine does not give the characteristic osazone, but an
amorphous precipitate which could not be induced to crystallize.
These tests are identical with those given by the solution of
starch after treatment with hydrogen peroxide and ferrous sulfate,
and upon this basis the conclusion is reached that glucose, and
possibly maltose, is formed in the course of the reaction only to be
further hydrolyzed and oxidized to form acids and free aldehyde.
By the use of ethyl alcohol, there may be precipitated from the
reaction mixture substances which have the properties of the
dextrins formed by the action of acids or enzymes upon starch
(Table III). By precipitating these substances while the solution
still produced a red color with iodine (i), there was obtained a
gummy residue which was quite soluble in water. Dialysis of this
solution for 10 days removed all cupric reducing power, but left a
solution which gave a deep red color with iodine and showed con-
siderable optical activity.
100 x 2.23
[ffl%,, = 1.0 x 1.15 = +193.8”

Hydrolysis of this substance gave a quantitative yield of glucose.

 W. R. Brown
TABLE III
Precipitation and Dialysis of Dextrins Precipitated from Reaction
Soluble starch
(Treated with HzOz and FeS04 until red color is produced with I,)
CtH,OH + N&l

i
(i) Residue (i, A) Filtrate
Clear gummy mass
Red color with I2

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Strong reduction (Benedict’s)
I Dextrorotatory

Clear Gatery solution

C2H60H + NaCl

(ii) Residue (ii, A) Filtrate

(Same as above, rotation (Combined with i, A,
and reduction less) alcohol removed)
Reduction
Dialyzed in cellophane Formic acid
I Free aldehyde
Acids
I No osazone

(f) Residual solution (v) Dialysate (iv) Residual solution

‘Rotation less Reduction No reduction
I Reduction less Dextrorotatory Less rotation
‘1 Red color with 12 1 No osazone Molisch +
(No osazone 1 No osazone

HCl (boiled) HCl (boiled) HCl (boiled)

I
Sugar solution Sugar solution Sugar solution
More reduction More reduction Reduction
i Less rotation Less rotation 1 Less rotation
(Glucosazone formed Glucosazone formed [Glucosazone formed
422 Starch Hydrolysis by H,O, and FeSO,

The concentrated diffusate (ii, Table III) from the dialysis of

the erythrodextrin was found to possess considerable optical
rotation and some reducing power.

[al::,, = 100 x 1.0 = +138.5”

1.0 x 0.794

l&90 = 100 X 0.963 = +122.4’

1.0 x 0.794

R (cupric reducing power) = 23.8 (maltose = 100)

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This is in fair agreement with the constants for amylotriose from
bacterial degradation of starch, given by Pringsheim (9).
[(Y]~& = +124.5”, R = 22.5 (maltose = 100)

Hydrolysis of this solution by dilute acid increased the reducing

power and decreased the optical rotation to that of a solution of
glucose, and from the hydrolyzed solution the characteristic
crystals of phenylglucosazone were obtained.
Precipitation of the original mixture, after the achromic point
had been reached, yielded a gummy precipitate (i, Table III)
as in the case of the erythrodextrin. This substance was soluble
in water but gave no color with iodine. Prolonged dialysis re-
moved all reducing power, but the solution still showed optical
activity.

l&,, = f,” ;‘;5” = +180.0°

Hydrolysis of this solution by acid caused a reduction of optical

rotation and an increase in the cupric reducing power until these
agreed with those of a solution of glucose. The diffusate (v)
from the dialysis of the achroodextrin was found to possess the
following constants.
100 X 0.81
r&:,, = = +52.53”
1 X 1.58

R = 4.1 (maltose = 100)

Hydrolysis of this solution increased the reducing power and de-

creased the optical rotation to some extent. This solution was
 W. R. Brown
undoubtedly a mixture of the oxidation products of glucose con-
taining a small amount of a substance of high optical rotation.

DISCUSSION

Hydrogen peroxide and ferrous sulfate react with starch to

produce a hydrolysis. The opalescence of the starch is lost, its
viscosity and optical rotation are reduced, its ability to reduce
alkaline copper is increased, and from the reaction there may be
isolated dextrins and the oxidation products of the simple sugars.

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With the exception of the further oxidation of the simple sugars
produced, these characteristics are identical with those produced
by enzymic or acid hydrolysis of starch. On this fact is based the
conclusion that the action of the system is a true hydrolysis,
analogous to that produced by the amylolytic enzymes.
The role of the metal in the reaction was adduced from several
facts. First, the presence of iron or similar metal appears to be
necessary for the hydrolysis. Although metal-free starch was not
used in the experiments, the rate of the reaction without added
metal was very slow, requiring several days to go to completion.
The dependence of the rate of hydrolysis upon the amount of
added iron (Table II) indicates that the complete removal of iron
would cause the hydrolysis to be immeasurably slow.
Iron has the ability to catalyze the decomposition of hydrogen
peroxide as does heat and alkalinity. The fact that the ability of
the metals to catalyze the hydrolysis of starch is not in the order
of their ability to catalyze the decomposition of the peroxide
indicates that the iron plays a part other than the mere liberation
of energy from the peroxide. The decomposition of the peroxide
by heat or alkalinity does not cause hydrolysis of the starch in a
measure to be expected from the amount of energy liberated.
The starch, peroxide, and iron must be in the same solution
for the hydrolysis to occur, indicating a loose chemical union.
That the union between the iron and starch is at the alcoholic
group seems unlikely, since such compounds are formed only in
alkaline solution. The logical point of attachment is through the
residual valences of the oxygen of the glucoside linkage of the
starch, for at this point the splitting occurs.
In the light of these facts, it seems that the iron (or other metal),
in addition to catalyzing the liberation of energy from the peroxide,
424 Starch Hydrolysis by H,Oz and FeS04

unites with the starch in a loose chemical union to pass the energy
of the peroxide decomposition into the starch molecule. The
energy level of the starch is thus raised, causing the starch to be
reactive.
The following theory of the mechanism of starch hydrolysis is
thus advanced: The iron atom forms an unstable combination
with the starch, possibly through the residual valences of the
oxygen of the glucoside linkage. The iron gives up its energy to
the starch molecule, thus raising the energy level of the starch

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molecule, and making it, more reactive. The iron, even in its
highest energy level, does not contain enough energy to split the
starch except, at a very slow rate. The decomposition of the per-
oxide produces large quantities of energy which is taken up to
form activated iron, and the energy of the iron is passed into the
starch molecule. The activated starch reacts with water and is
hydrolyzed. Enough energy is put into the starch molecule to
cause a quite rapid hydrolysis. The iron, upon giving up the
energy to the starch molecule, reverts to a lower energy level, is
again activated by the peroxide, and in turn passes this energy to
another molecule of starch. The reaction is a true catalysis, since
the iron left is available for many transfers of energy. This is in
line with Warburg’s (10) suggestion that FeIV is the form of
iron in its active state.
Fe” -+ Fe”’ --+ Fe’”

1,

dextrins
Fe’” + starch + Hz0 ---+ Fel’ + glucose
1 oxidation products

The peroxide-iron-starch system appears to be identical with the

hydrolytic enzymes except for the fact that the supply of available
energy is limited to the amount of peroxide present, and the
system may be called an artificial enzyme.

SUMMARY

The action of hydrogen peroxide and ferrous sulfate upon

starch is a hydrolysis, producing in the course of the reaction
dextrins, sugars of high molecular weight, and simple sugars. The
reaction is analogous to that produced by amylase, differing only
 W. R. Brown 425
in the fact that the simple sugars produced are further hydrolyzed
and oxidized to acids and aldehydes. The reaction appears to be
a true catalysis, the iron acting to transfer energy from the per-
oxide breakdown to the starch molecule, thus raising the energy
level of the starch and causing it to be reactive.

I wish to take this opportunity to express my sincere gratitude

and appreciation to Dr. Albert P. Mathews for advice and assist-
ance during the course of this study. To Mr. Charles G. Merrell

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I am deeply indebted for the donation of the William S. Merrell
Fellowship.

BIBLIOGRAPHY

1. Mathews, A. P., in Cowdry, E. V., General cytology, Chicago, 15 (1924).

2. Hill, E. S., J. Biol. Chem., 96,197 (1932).
3. Boyd, M. J., J. Biol. Chem., 103,249 (1933).
4. Sigal, A., Thesis, University of Cincinnati (1932).
5. Gatin-Gruzewska, M., Compt. rend. Sot. biol., 68, 1084 (1910).
6. Gerber, C., Compt. rend. Sot. biol., 72,1002 (1912).
7. Durieux, O., Bull. Sot. chim. Belgique, 27,90 (1913).
8. Omori, T., J. Biochem., Japan, 14, 339 (1931); 16,483 (1932).
9. Pringsheim, H., Ber. them. Ges., &I’,1581 (1924).
10. Warburg, O., Science, 61,575 (1925).